Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof

ABSTRACT

The  F. necrophorum  gene expressing leukotoxin was sequenced and cloned. The leukotoxin open reading frame (lktA) is part of a multi-gene operon containing 9,726 bp, and encoding a protein containing 3,241 amino acids with an overall molecular weight of 335,956 daltons. The protein encoded by the gene was truncated into five polypeptides having overlapping regions by truncating the full length gene into five different sections and amplifying, expressing, and recovering the protein encoded by each of these sections. Additionally, a region upstream of the gene was sequenced and the polypeptide encoded by that nucleotide sequence was purified and isolated. These polypeptides along with the full length protein are then tested to determine their immunogenicity and protective immunity in comparison to the efficacy of immunization conferred by inactivated native leukotoxin in  F. necrophorum  culture supernatant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.09/558,257, filed Apr. 25, 2000, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is concerned with methods of cloning andexpressing the leukotoxin gene from Fusobacterium necrophorum (F.necrophorum), sequencing and characterizing the leukotoxin proteinexpressed by this gene, truncating the gene into a series of nucleotidesequences, amplifying these sequences, expressing and recovering thepolypeptides encoded by the nucleotide sequences, and utilizing theprotein and the polypeptides in recombinant vaccines in order to confereffective immunity against infection caused by the production ofleukotoxin by F. necrophorum. More particularly, it is concerned withproduction of an inactivated recombinant leukotoxin vaccine generated byamplifying five leukotoxin gene fragments and one upstream regionthrough PCR, digesting the nucleotide sequences encoded by the genefragments with restriction enzymes, expressing the polypeptide sequencescoded by the nucleotide sequences through an expression vector,recovering these proteins as five truncated leukotoxin proteins (orpolypeptides), purifying these proteins (or polypeptides) to apparenthomogeneity, with or without inactivation of the truncated and fulllength proteins, and combining the inactivated recombinant leukotoxinswith adjuvants.

2. Description of the Prior Art

Liver abscesses in feed lot cattle are a serious economic problem,causing condemnation of over 3 million livers and an estimated loss of$15 million annually in the United States. This estimate is basedprimarily on condemnation of liver and other organs, and does notinclude economic losses stemming from reduced feed intake, reduced feedefficiencies, decreased carcass dressing percentage and lowered weightgains. A number of studies have confirmed that cattle with abscessedlivers gain less (average 4-5%) and have reduced feed efficiencies(average 7%) compared with cattle having healthy livers. The averageincidence of abscessed liver in grain-fed cattle approximates 25-30%. Toa lesser extent, liver abscesses in sheep and goats are also an economicproblem.

F. necrophorum is a gram-negative, rod-shaped, nonsporeforming,nonmotile, strictly anaerobic and pleomorphic organism. Morphologically,the organism varies from short rods to filamentous with pointed androunded ends. Cell lengths range from coccoid bodies of 0.5-0.7 μmdiameter to filaments over 100 μm. Surface colonies are 1-2 mm indiameter, circular, transparent to opaque, and with some strainsproducing α or β hemolysis. The organism ferments glucose, fructose andmaltose only weakly with final pH around 5.0-6.3. It ferments lactate toacetate, propionate, and butyrate. Butyrate is the major product fromlactate fermentation. Indole is produced from peptone. F. necrophorumhas been isolated from the normal flora in the oral cavity,gastrointestinal cavity, and genitourinary tract of humans and animals.The organism is also known to survive in the soil.

F. necrophorum is a normal inhabitant of the gastrointestinal tracts ofanimals and humans. Virulence factors and pathogenic mechanisms thatcontribute to the transition of this otherwise commensal organism to apathogen are poorly understood. A leukotoxin, endotoxin, hemolysin,hemagglutinin, and several enzymes such as deoxyribonuclease andproteases have been suggested as possible virulence factors. However,several studies implicate leukotoxin, a protein cytotoxic to ruminantpolymorphonuclear cells, as the major virulence factor. The importanceof leukotoxin as a virulence factor in F. necrophorum infections isindicated by a correlation between toxin production and ability toinduce abscesses in laboratory animals, an inability ofnonleukotoxin-producing strains to induce foot abscesses in cattlefollowing intradermal inoculation, and a relationship betweenantileukotoxin antibody titers and protection against infection inexperimental challenge studies.

F. necrophorum is an opportunistic pathogen that is the primaryetiologic agent of liver abscesses in ruminant animals. (Scanlan, etal., (1983) Bovine rumenitis-liver abscess complex: a bacteriologicalreview. Cornell Vet. 73:288-297; Nagaraja, T. G. et al., (1998) Liverabscesses in feedlot cattle: A review. J. Anim. Sci., 76:287-298; andTan, et al., (1996) Fusobacterium necrophorum infections: virulencefactors pathogenic mechanism and control measures. Vet. Res. Comm.,20:113-140). The organism has been recognized as an animal and humanpathogen since the late 1800s, and is associated as a primary orsecondary etiologic agent with numerous necrotic disease conditions indomestic and wild animals. In addition to liver abscesses, the organismis also the primary etiologic agent of foot rot, foot abscesses, calfdiphtheria, and is frequently isolated from cases of mastitis, metritis,and necrotic lesions of the oral cavity.

Liver abscesses in cattle are part of a disease complex where theabscessation is secondary to primary foci of infection in the rumenepithelium. The pathogenesis can be summarized as follows: (1) ruminallesions are induced by acidosis that follows rapid change in diet fromhigh-roughage to high grain, prolonged feeding of high grain diet, oroccasionally by foreign body penetration of the rumen epithelium; (2)bacteria present in the rumen invade the epithelium and form focalabscesses in the rumen wall; and (3) bacteria enter the portalcirculation, and are carried to the liver where they localize in theparenchyma with subsequent abscess formation.

The ability of F. necrophorum to establish in the liver is attributed tothe production of a toxin which is a secreted protein of high molecularweight active against leukocytes from ruminants called leukotoxin (orleucocidin). The toxin is a soluble extracellular protein that iscytotoxic to neutrophils, macrophages, hepatocytes, and ruminal cells.The leukotoxin protects against phagocytosis and is believed to aid inthe establishment of F. necrophorum in the liver by directly impairingthe normal defense mechanism and indirectly by the damage caused bycytolytic products released from neutrophils and macrophages to thehepatic cells. Therefore, the leukotoxin elaborated from F. necrophorumplays a critical role in F. necrophorum infection of the liver and isbelieved to be the primary virulence factor in the pathogenesis of liverabscesses (Tan et al., 1996).

Four biotypes (A, B, AB and C) of F. necrophorum have been described.(Langworth, (1977) Fusobacterium necrophorum: its characteristics androle as an animal pathogen. Bacteriol. Rev. 41:373-390) Biotype A, mostfrequently isolated from liver abscesses, is more pathogenic thanbiotype B, which predominates in ruminal wall abscesses. Biotypes AB andC are rarely isolated in liver abcesses (Berg, et al., (1982) Studies ofFusobacterium necrophorum from bovine hepatic abscesses: Biotypes,quantitation, virulence, and antibiotic susceptibility. Am. J. Vet. Res.43:1580-1586), and biotype A has pathogenicity intermediate that ofbiotypes A and B while biotype C is non-pathogenic. (Shinjo, et al.,(1990) Recognition of biovar C of Fusobacterium necrophorum (flugge)Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom.rev. (ex prevot 1940) Int. J. Sys. Bacteriol. 41:395-397) Biotypes A andB, the most frequent types encountered in liver abscesses, have beenassigned subspecies status: subsp. necrophorum and subsp. funduliforme,respectively (Shinjo et al., 1990). The subsp. necrophorum is morevirulent, produces more leukotoxin and hemagglutinin, and is morefrequently isolated from cattle liver abscesses than the subsp.funduliforme. Virulence factors and pathogenic mechanisms contributingto the formation of liver abscesses by F. necrophorum are poorlyunderstood (Tan et al., 1996). However, several studies implicateleukotoxin to be a major virulence factor (Emery, et al., (1986)Generation of immunity against Fusobacterium necrophorum in miceinoculated with extracts containing leukotoxin. Vet. Microbiol.12:255-268; Tan et al., 1996). The importance of leukotoxin is evidencedby correlation between toxin production and ability to induce abscessesin laboratory animals (Coyle-Dennis, et al., (1979) Correlation betweenleukocidin production and virulence of two isolates of Fusobacteriumnecrophorum. Am. J. Vet. Res. 40:274-276; Emery and Vaughn, 1986),inability of nonleukotoxin-producing strains to induce foot abscesses incattle following intradermal inoculation (Emery, et al., (1985) Culturecharacteristics and virulence of strains of Fusobacterium necrophorumisolated from feet of cattle and sheep. Australian Vet. J. 62:43-46) andrelationship between antileukotoxin antibody titers and protection inexperimental challenge studies (Saginala, et al., (1996a) The serumneutralizing antibody response in cattle to Fusobacterium necrophorumleukotoxoid and possible protection against experimentally inducedhepatic abscesses. Vet. Res. Comm., 20:493-504; Saginala, et al.,(1996b) The serum neutralizing antibody response and protection againstexperimentally induced liver abscesses in steers vaccinated withFusobacterium necrophorum. Am. J. Vet Res., 57:483-488; and Shinjo, etal., (1991) Proposal of two subspecies of Fusobacterium necrophorum(Flugge) Moore and Holdeman: Fusobacterium necrophorum subsp.necrophorum subsp. nov., nom. rev. (ex Flugge 1886), and Fusobacteriumnecrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hall 1898).Int. J. Sys. Bacteriol. 41:395-397).

Several investigators have attempted to induce protective immunityagainst F. necrophorum by using a variety of antigenic components. Theresults of such attempts have varied from ineffectual to significantprotection. Clark et al. reported that cattle injected with F.necrophorum culture supernatant containing leukotoxin had a lowincidence of foot rot caused by F. necrophorum. (Clark, et al. (1986),Studies into immunization of cattle against interdigitalnecrobacillosis. Aust. Vet. J. 63:107-110) Cell-free culture supernatantof a high leukotoxin producing strain of F. necrophorum (Tan et al.,(1992) Factors affecting leukotoxin activity of F. necrophorum. Vet.Microbiol. 33:15-28), mixed with an adjuvant, was shown to elicit a highantileukotoxin antibody titer when injected in steers and providedsignificant protection to experimentally induced liver abscesses(Saginala et al., 1996a, b; 1997). F. necrophorum bacterin was used asan agent for immunizing cattle and sheep against liver necrosis as shownin EPO Application No. 460480 of Dec. 11, 1991 (the teachings of whichare incorporated herein by reference). Specifically, virulent F.necrophorum isolates are inactivated using β-propiolactone, followed byaddition of adjuvants. In addition, Abe et al., Infection and Immunity,13:1473-1478, 1976 grew F. necrophorum for 48 hours. Cells were obtainedby centrifuging, washing three times with saline, and were inactivatedwith formalin (0.4% in saline). The inactivated cells were then injectedinto mice to induce immunity. Two weeks after the last boosterinjection, each mouse was challenged with viable cells of F.necrophorum. The mice immunized with killed cells and challenged withlive cells had no detectable bacteria in the liver, lung or spleen forup to 28 days. It was concluded that immunization of mice withformalin-killed F. necrophorum conferred protection against infection.Garcia et al., (Canadian J. Comp. Med, 38:222-226, 1974), conductedfield trials to evaluate the efficacy of alum-precipitated toxoids of F.necrophorum. The vaccine preparation consisted of washed cells (unlikelyto contain leukotoxin) that were ruptured by sonication. The mostpromising result was achieved with the injection of 15.5 mg protein ofcytoplasmic toxoid. In this group, the incidents of liver abscesses wasreduced to 10% from an average 35% in the control group. Emery et al.,Vet. Microbiol., 12:255-268, 1986, prepared material by gel filtrationof 18-hour culture supernate of F. necrophorum. This elicitedsignificant immunity against challenge by with viable F. necrophorum.The injected preparation contained endotoxin and the majority of theleukotoxic activity. U.S. Pat. No. 5,455,034 (the teachings of which areincorporated herein by reference) by Nagaraja et al. disclosed thatprevention of leukotoxin production (or inhibition of its activity) inimmunized animals prevents the establishment of F. necrophoruminfection. Thus, immunization of the animals against F. necrophorumleukotoxin, so that the animals' white blood cells or tissue macrophagesmay phagocytize the bacteria, presented a way to prevent diseasesassociated with F. necrophorum infection, e.g., liver abscesses incattle and sheep, and foot rot in cattle. In order to produce such aleukotoxoid vaccine, the F. necrophorum bacteria was cultured in away toenhance the elaboration of leukotoxin in the supernate. Thereupon,bacterial growth and leukotoxin elaboration was terminated, and avaccine prepared by inactivating at least the leukotoxin-containingsupernate. In more detail, the leukotoxin elaboration method of the '034patent involved first forming a culture of F. necrophorum bacteria ingrowth media, and thereafter causing the bacteria to grow in the cultureand to simultaneously elaborate leukotoxin in the supernate. At the endof the culturing step, i.e., at the end of the selected culture timewithin the range of from about 4-10 hours, the bacterial growth andleukotoxin elaboration were terminated, and the leukotoxoid vaccine wasprepared. This involved first separating the leukotoxin-containingsupernate from the bacteria, followed by inactivation through use offormalin, β-propiolactone, heat, radiation or any other known method ofinactivation. Alternately, the entire culture could be inactivated toform the vaccine.

Presently, the control of liver abscesses is with the use ofantimicrobial feed additives. Antimicrobial compounds reduce theincidence of liver abscesses but do not eliminate the problem (Nagarajaet al., 1998). Therefore, an effective vaccine would be highly desirableto the feedlot industry. The vaccine approach also would alleviatepublic health concerns associated with the use of subtherapeutic levelsof antibiotics in the feed. Because studies have indicated thatantileukotoxin immunity reduces the incidence of hepatic abscesses andinterdigital necrobacillosis (Garcia et al., 1974; Clark et al., 1986;Saginala et al., 1996a, b; 1997), the development of a recombinantleukotoxin vaccine will be of great value in the control of hepatic andinterdigital necrobacillosis in cattle.

SUMMARY OF THE INVENTION

In order to better define the molecular nature of the F. necrophorumleukotoxin, and as a first step toward determining its specific role inthe virulence of this bacterium, the leukotoxin gene was isolated, itsnucleotide sequence determined, and the recombinant leukotoxin wasexpressed in E. coli.

The leukotoxin open reading frame (lktA) is part of a multi-gene operoncontaining 9,726 bp, and encoding a protein containing 3,241 amino acidswith an overall molecular weight of 335,956 daltons. F. necrophorumleukotoxin is highly unstable as evidenced by western blot analysis ofnative leukotoxin (culture supernatant, sephadex gel or affinitypurified) (FIG. 1). In this Figure, lane 1 contains whole cell lysate ofE. coli cells expressing full-length recombinant leukotoxin, lane 2contains Immuno-affinity purified native leukotoxin, lane 3 containsSephadex gel purified leukotoxin, and lane 4 contains culturesupernatant from F. necrophorum concentrated 60 times. The blots wereprobed with polyclonal antiserum raised in rabbits against affinitypurified native leukotoxin. Because of the apparent instability of thefull-length recombinant leukotoxin protein, the protein encoded by thegene was truncated into five recombinant polypeptides (or proteinfragments, BSBSE, SX, GAS, SH and FINAL) having overlapping regions bytruncating the full length gene into five different sections andamplifying, expressing in E. coli, and recovering the protein orpolypeptide encoded by each of these sections. These polypeptides alongwith the full length protein are then tested to determine theirimmunogenicity and protective immunity in comparison to the efficacy ofimmunization conferred by inactivated native leukotoxin in F.necrophorum culture supernatant.

Specifically, the chromosomal DNA was extracted from F. necrophorum andpartially digested by restriction endonucleases prior to beingsize-fractionated by sucrose gradient centrifugation. The 10-12 kbfragments were then ligated into a BamHI digested, dephosphorylated λZAPexpression vector. Recombinant phages were infected into Escherichiacoli and plated onto agar plates. Plaque lifts were performed (withpolyclonal antiserum raised in rabbits against affinity purifiedleukotoxin) using an immunoscreening kit. Six immunoreactive recombinantphages were identified and denominated as clones 816, 611, 513, 911,101, and 103. These clones were plaque-purified three times to ensurepurity, phagemids rescued, and anti-leukotoxin immunoreactivity of theencoded proteins was confirmed. This immunoreactivity verified that theclones represented native leukotoxin F. necrophorum.

Expression of a polypeptide encoded by the 3.5 kb from the 5′ end of thelktA caused immediate cessation of the growth and lysis of E. coli hostcells suggesting that regions of leukotoxin could be toxic to E. coli.Of course, the objective was to create overlapping gene truncationsextending over the entire lktA ORF so that the resulting polypeptideproducts are small and relatively stable on expression, but are largeenough to be immunogenic. Also, the effectiveness of various recombinanttruncated leukotoxin polypeptides alone or in combinations as immunogensand evaluated protective immunity against challenge with F. necrophorumin mice was investigated. The use of mice as an experimental model forF. necrophorum infection in cattle is well established (Abe et al.,1976; Conion et al., 1977; Smith et al., 1989; Garcia and McKay, 1978;Emery and Vaughan, 1986). Extension of the patterns of immunity andinfection to cattle has shown that mice can be a valuable model toevaluate the immunogenicity and protection provided by various F.necrophorum fractions (Garcia et al., 1975; Garcia and McKay, 1978).Studies have also indicated that strains of F. necrophorum that arepathogenic in domestic animals, frequently are pathogenic in micesuggesting necrobacillosis as a disease is similar among these speciesof animals (Smith and Thornton, 1993).

The nucleotide sequence of the full length version of the gene isdesignated as SEQ ID No. 8 and the nucleotide sequences of the fivetruncations of the full length gene are designated as BSBSE (SEQ ID No.9), SX (SEQ ID No. 10), GAS (SEQ ID No. 11), SH (SEQ ID No. 12), andFINAL (SEQ ID No. 13). Additionally, the nucleotide sequence of theupstream region of the full length gene is designated UPS (SEQ ID No.14). The amino acid sequence of the full length protein encoded by theF. necrophorum gene is designated as SEQ ID No. 1 and the amino acidsequences of the truncated protein fragments respectively encoded byBSBSE, SX, GAS, SH and FINAL are designated as SEQ ID No. 2, SEQ ID No.3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6. In the case of UPS, thepolypeptide or truncated protein fragment encoded for by UPS isdesignated as SEQ ID No. 7. Finally, SEQ ID No. 15 is the fall lengthgene sequence along with contiguous sequences.

Truncated recombinant polypeptides were purified by nickel affinitychromatography, and injected into rabbits to raise polyclonal antisera.Antibodies raised against two of the five polypeptides (BSBSE and GAS)neutralized the toxicity of F. necrophorum leukotoxin against bovineneutrophils. The effectiveness of the purified truncated polypeptides toinduce a protective immunity was determined by injecting thepolypeptides, individually or in mixtures, homogenized with Ribiadjuvant in mice, followed by experimental challenge with F.necrophorum. Two polypeptides (BSBSE and SH) induced significantprotection in mice against F. necrophorum infection and the extent ofprotection was greater than the full-length native leukotoxin orinactivated culture supernatant. The study provided further credence tothe importance of leukotoxin as the major virulence factor of F.necrophorum and the protein carries a domain(s) or epitope(s) thatinduces protective immunity against experimental infection.

The DNA and deduced amino acid sequences were compared with sequences inGenbank but no significant similarities (no sequences having greaterthan 22% sequence identity) were found. Thus, the F. necrophorumleukotoxin appears to be distinct from all known leukotoxins andRTX-type toxins. When the deduced amino acid sequence of the lktA regionwas subjected to the Kyte-Doolittle hydropathy analysis (FIG. 3), 14sites of sufficient length and hydrophobic character to be potentialmembrane spanning regions, were found. Upstream to the leukotoxin ORF isan open reading frame of at least 1.4 kb in length, which is in the sameorientation. It encodes a protein that has significant sequencesimilarity (21% or 62 out of 283 residues) to the heme-hemopexinutilization protein (UxuB) of Haemophilus infuenzae.

Bacterial leukotoxins and cytotoxins generally have molecular masses ofless than 200 kDa. This includes characterized leukotoxins ofPasteurella hemolytica (104,000 kDa; 10), Staphylococcus aureus(38,000+32,000 kDa; 20), or Actinomyces actinomycetecomitans (114,000kDa; 15) or other pore-forming toxins of gram-negative bacteria(103,000to 198,000 kDa; 30). However, leukotoxin secreted by F.necrophorum was shown to be approximately 300 kDa in size based onsephadex column purification and SDS-PAGE analyses.

As used herein, the following definitions will apply: “SequenceIdentity” as it is known in the art refers to a relationship between twoor more polypeptide sequences or two or more polynucleotide sequences,namely a reference sequence and a given sequence to be compared with thereference sequence. Sequence identity is determined by comparing thegiven sequence to the reference sequence after the sequences have beenoptimally aligned to produce the highest degree of sequence similarity,as determined by the match between strings of such sequences. Upon suchalignment, sequence identity is ascertained on a position-by-positionbasis, e.g., the sequences are “identical” at a particular position ifat that position, the nucleotides or amino acid residues are identical.The total number of such position identities is then divided by thetotal number of nucleotides or residues in the reference sequence togive % sequence identity. Sequence identity can be readily calculated byknown methods, including but not limited to, those described inComputational Molecular Biology, Lesk, A. N., ed., Oxford UniversityPress, New York (1988), Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); Sequence Analysis in Molecular Biology, vonHeinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov,M. et al., eds., M. Stockton Press, New York (1991); and Carillo, H., etal. Applied Math., 48:1073 (1988), the teachings of which areincorporated herein by reference. Preferred methods to determine thesequence identity are designed to give the largest match between thesequences tested. Methods to determine sequence identity are codified inpublicly available computer programs which determine sequence identitybetween given sequences. Examples of such programs include, but are notlimited to, the GCG program package (Devereux, J., et al., Nucleic AcidsResearch, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F.et al., J. Molec. Biol.,215:403-410(1990). The BLASTX program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J.Molec. Biol., 215:403-410(1990), the teachings of which are incorporatedherein by reference). These programs optimally align sequences usingdefault gap weights in order to produce the highest level of sequenceidentity between the given and reference sequences. As an illustration,by a polynucleotide having a nucleotide sequence having at least, forexample, 95% “sequence identity” to a reference nucleotide sequence, itis intended that the nucleotide sequence of the given polynucleotide isidentical to the reference sequence except that the given polynucleotidesequence may include up to 5 point mutations per each 100 nucleotides ofthe reference nucleotide sequence. In other words, in a polynucleotidehaving a nucleotide sequence having at least 95% identity relative tothe reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence maybe deleted or substituted with another nucleotide,or a number of nucleotides up to 5% of the total nucleotides in thereference sequence maybe inserted into the reference sequence. Thesemutations of the reference sequence may occur at the 5′ or 3′ terminalpositions of the reference nucleotide sequence or anywhere between thoseterminal positions, interspersed either individually among nucleotidesin the reference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having a given aminoacid sequence having at least, for example,95% sequence identity to areference amino acid sequence, it is intended that the given amino acidsequence of the polypeptide is identical to the reference sequenceexcept that the given polypeptide sequence may include up to 5 aminoacid alterations per each 100 amino acids of the reference amino acidsequence. In other words, to obtain a given polypeptide sequence havingat least 95% sequence identity with a reference amino acid sequence, upto 5% of the amino acid residues in the reference sequence maybe deletedor substituted with another amino acid, or a number of amino acids up to5% of the total number of amino acid residues in the reference sequencemay be inserted into the reference sequence. These alterations of thereference sequence may occur at the amino or the carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in the one or more contiguous groups withinthe reference sequence. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. However,conservative substitutions are not included as a match when determiningsequence identity.

Similarly, “sequence homology”, as used herein, also refers to a methodof determining the relatedness of two sequences. To determine sequencehomology, two or more sequences are optimally aligned as describedabove, and gaps are introduced if necessary. However, in contrast to“sequence identity”, conservative amino acid substitutions are countedas a match when determining sequence homology. In other words, to obtaina polypeptide or polynucleotide having 95% sequence homology with areference sequence, 95% of the amino acid residues or nucleotides in thereference sequence must match or comprise a conservative substitutionwith another amino acid or nucleotide, or a number of amino acids ornucleotides up to 5% of the total amino acid residues or nucleotides,not including conservative substitutions, in the reference sequencemaybe inserted into the reference sequence.

A “conservative substitution” refers to the substitution of an aminoacid residue or nucleotide with another amino acid residue or nucleotidehaving similar characteristics or properties including size, charge,hydrophobicity, etc., such that the overall functionality does notchange significantly.

Isolated” means altered “by the hand of man” from its natural state.,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Finally, all references and teachings cited herein which havenot been expressly incorporated by reference are hereby incorporated byreference.

Preferably, sequences having at least about 50% sequence homology or atleast about 60% sequence identity with any of SEQ ID Nos. 1-15 are usedfor purposes of the present invention. More preferably, sequences havingat least about 60% sequence homology or at least about 70% sequenceidentity are used for purposes of the present invention. Still morepreferably, sequences having at least about 75% sequence homology or atleast about 85% sequence identity are used for purposes of the presentinvention. Even more preferably, sequences having at least about 87%sequence homology or at least about 92% sequence identity are used forpurposes of the present invention. Most preferably, sequences having atleast about 95% sequence homology or at least about 98% sequenceidentity are used for purposes of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot assay of native and recombinant leukotoxins.

FIG. 2 is an illustration of the fall length F. necrophorum gene and amap of the truncated regions of the genes and the expression clonesencoded by the truncated regions;

FIG. 3 is a Kyte-Doolittle hydropathy plot of the leukotoxin from F.necrophorum;

FIG. 4 is an illustration of the Southern Hybridization pattern of thechromosomal DNA of F. necrophorum with inserts from clones 513, 611,816, 911, and 101;

FIG. 5 is a Kyte-Doolittle hydropathy plots of deduced amino acidsequences from the F. necrophorum leukotoxin gene wherein the linesabove the plot correspond to the regions of the five truncated LktApolypeptides (BSBSE, SX, GAS, SH, and FINAL).

FIG. 6 is an illustration of the leukotoxin locus of F. necrophorum.

FIG. 7a is a Western blot analysis of truncated forms of purifiedrecombinant leukotoxin protein probed with polyclonal antileukotoxinantiserum.

FIG. 7b is a Western blot analysis of truncated forms of purifiedrecombinant leukotoxin protein probed with monoclonal antibody F7B10

FIG. 7c is a Western blot of whole-cell lysates from E. coli clonesexpressing full-length recombinant leukotoxin probed with the monoclonalanti-leukotoxin antibody.

FIG. 8 is a graph illustrating the evaluation of leukotoxic activity byflow cytometry.

FIG. 9 is graph illustrating the toxicity of the recombinant leukotoxinand the truncated polypeptides by flow cytometry.

FIG. 10 is an illustration of the hybridization patterns of radiolabeled lktA with Southern blotted HaeIII digested restriction fragmentsof genomic DNAs from F. necrophorum subsp. necrophorum isolates fromliver abscesses;

FIG. 11 is an illustration of the expression clones for the truncatedproteins designated UPS, BSBSE, SX, GAS, SH, and FINAL.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following examples set forth preferred embodiments of the presentinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

EXAMPLE 1 Cloning of the Leukotoxin Encoding F. necrophorum Gene

Chromosomal DNA, extracted from Fusobacterium necrophorum subsp.necrophorum, strain A25 (Hull et al., 1981, Construction and expressionof recombinant plasmids encoding type 1 or D-mannose-resistant pili froma urinary tract infection Escherichia coli isolate. Infect. Immun.33:933-938.), was partially digested with the restriction endonucleaseSau3AI, and size-fractionated by sucrose gradient centrifugation(Baxter-Gabbard, 1972, A simple method for the large scale preparationof sucrose gradients. FEBS. Lett. 20117-119). The 10-12 kb DNA fragmentswere ligated in to BamHI-digested, dephosphorylated λZAP Express vector,packaged into lambda phage head and tail protein components (Stratagene,La Jolla, Calif.), and recombinant phages were infected into Escherichiacoli XL1-Blue MRF′ and plated onto agar plates. Plaque lifts wereperformed (with polyclonal antiserum raised in rabbits against affinitypurified leukotoxin) using the Pico-blue immunoscreening kit(Stratagene, La Jolla, Calif.). Six immunoreactive recombinant phageswere identified (816, 611, 513, 911, 101, and 103; FIG. 2). These cloneswere plaque-purified three times to ensure purity, and anti-leukotoxinimmunoreactivity of the proteins was confirmed.

Characterization of the Leukotoxin Gene Excision of the Cloned DNAInsert into a Phagemid Vector

The λZAP Express vector is composed of a plasmid, designated pBK-CMV,which flanks the cloned insert DNA and which can be readily excised inorder to obtain a phagemid that contains the cloned insert DNA.Therefore, a recombinant phagemid containing cloned F. necrophorum DNAinsert was obtained by simultaneously infecting E. coli XLOLR withExAssist helper phage and the recombinant phage (containing the clonedF. necrophorum DNA) according to the manufacturers instructions(Stratagene, La Jolla, Calif.). Once the recombinant plasmid wasrecovered, the presence of the DNA insert was confirmed by restrictionendonuclease digestion and agarose gel electrophoresis.

Physical Mapping of the F. necrophorum DNA Inserts

Restriction enzyme digestion and mapping of the recombinant phagemid wasperformed (Sambrook et al., 1989, Molecular cloning: a laboratorymanual. Cold spring harbor laboratory, Cold Spring Harbor, N.Y.).Combinations of the restriction enzymes SacI, SalI, SpeI, BamHI, EcoRI,HindIII, PstI, DraI, XbaI, HaeIII, BglII, SmaI, and KpnI were used forrestriction enzyme mapping since single sites for these enzymes exist inthe multiple cloning site of pBK-CMV. Insert DNA from all the siximmunoreactive clones contained EcoRI, PstI, HindIII, DraI, HaeIII andBglII sites but not sites for Sac I, SmaI, SalI, XbaI, KpnI or BamHI.

Hybridization of the Cloned DNA Fragments with F. necrophorumChromosomal DNA

Southern hybridization (Southern, 1975, Detection of specific sequencesamong DNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503) experiments were performed to confirm that the cloned DNAencoding the putative leukotoxin gene originated from F. necrophorumstrain A25. Inserts from clones 513, 611, 816 and 911 were separatedfrom the vector sequence by agarose gel electrophoresis of DNA digestedwith restriction enzymes SalI and XbaI. The insert DNA was used as aprobe to hybridize to chromosomal DNA of F. necrophorum digested withEcoRI, EcoRV, HaeIII, and HindIII. A negative control, E. coli DH5α DNA,was digested with EcoRV. The Southern hybridization patterns includedcommon DNA fragments indicating that the six clones carried overlappinginserts (FIG. 4). FIG. 2 illustrates the overlapping of each of the siximmunoreactive clones designated 816, 611, 513, 911, 101, and 103. Theexpression clones for truncated peptides are designated UPS, BSBSE, SX,GAS, SH, and FINAL while the numbers in parentheses indicate the size inkilo-bases of each insert. The overlaps illustrated in FIG. 2 werefurther confirmed by sequence analysis.

DNA Sequence Analysis of the F. necrophorum DNA Inserts

Subclones of the cloned insert DNAs were constructed based on therestriction enzyme map of the cloned insert. Plasmid DNA was isolatedfrom the resulting subclones (Bimboim and Doly, 1979, A rapid alkalineextraction procedure for screening recombinant plasmid DNA. Nucleicacids Res. 7:1513) and subjected to DNA sequence analysis using theSanger dideoxy chain termination method (Sanger et al., 1977, DNAsequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci.74:5463-5467) using vector based primers. Additional sequence data wereobtained by creating deletion clones utilizing restriction endonucleasesites discovered in the preliminary sequencing or by sequencing usingprimers derived from the sequenced DNA.

A total of 9.3 kb of the leukotoxin chromosomal region was cloned andsequenced. A single large open reading frame (designated lktA) is commonto each of the immunoreactive clones. The ORF is preceded by a ribosomebinding site (RBS) sequence (AAGGGGGT). Eight base pairs following theRBS sequence is a start codon (the ninth base pair) for the open-readingframe, which is approximately 8 kb in length. The stop codon of lktA wasnot found in this region. Therefore, the downstream sequences wereextended by inverse PCR amplification, followed by cloning andsequencing of the amplified region.

Extension of the lktA Open Reading Frame Using Inverse PCR

Chromosomal DNA from F. necrophorum strain A25 was digested withrestriction endonucleases TaqI, EcoRI, DdeI, or Sau3AI individually.After complete digestion of the chromosomal DNA with any one of theseenzymes, the products were extracted with phenol and chloroform, andethanol precipitated. Under dilute conditions (100 μl final volume) 200ng of digested DNA was self-ligated using T4DNA ligase at 16 C overnight(Ochman et al., 1990, Amplification of flanking sequences by inversePCR. In: M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White(eds); PCR protocols; A guide to methods and applications. Acad. Press,Inc. Harcourt Brace Jovanovich, publishers, Sandiego, 219-227). LigatedDNA was phenol and chloroform extracted, ethanol precipitated andreconstituted in 10 μl of nuclease free water. Two microliters of theligated DNA were used as template for PCR reaction with forward andreverse primers designed based on the sequence already known to us fromprevious sequencing reactions. Amplified products were cloned in the pCR2.1 plasmid vector (Invitrogen) and sequenced using vector specificsequences. Sequencing six consecutive inverse PCR products enabled us toidentify the stop codon for leukotoxin gene and the presence of anotherORF downstream of lktA.

The entire leukotoxin gene was amplified using heat-stable DNApolymerase (ExTaq) as two fragments using F. necrophorum strain A25chromosomal DNA as the template. The 5′4.3 kb of the lktA open-readingframe encoding the N-terminal half of the leukotoxin, and the 3′5.4 kbrepresenting the C-terminal half of the leukotoxin protein. Making useof the unique Nhe I site present at this location (4.3 kb from the startcodon), the leukotoxin gene was joined together to give the giant 9.726kb ORF. The entire leukotoxin gene was cloned into the modified variant(with coding sequence for six histidine residues in the N-terminus ofthe expressed protein) of the expression vector pET 14b (Novagen Corp.Madison, Wis.). This T7 polymerase based system should enhanceexpression of toxic proteins, without damage to the host cell E. coli.

EXAMPLE 2 Preparation of Polyclonal Antileukotoxin Antiserum

Leukotoxin from F. necrophorum subsp. necrophorum strain A25 waspurified using an immunoaffinity column containing antileukotoxinmonoclonal antibody, F7B10 (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa,J. J. Staats. 1994. Purification and quantification of Fusobacteriumnecrophorum leukotoxin using monoclonal antibodies. Vet. Microbiol.42:121-133.). Affinity-purified native leukotoxin (0.5 mg) in 100 μl ofPBS was homogenized with an equal volume of Freund's complete adjuvantand injected intramuscularly in rabbits. A booster dose was given on day21 with 0.5 mg of native toxin in 100 μl of PBS homogenized with anequal volume of Freund's incomplete adjuvant. Serum samples werecollected on day 42. Naturally occurring rabbit antibodies that react toE. coli proteins were removed from the antisera as follows. Cell pelletsof E. coli XL1-Blue MRF′ host cells grown overnight in Luria broth weresonicated in PBS and centrifuged to remove cellular debris, and thesupernatant was incubated with 100 mm diameter nitrocellulose membranesat 37° C. for 3 hours. The nitrocellulose membranes were then washedtwice in PBS-T (0.05% Tween 20 in PBS [pH 7.2]), blocked in 2% BSA, andwashed three times again in PBS-T. Two ml of rabbit antileukotoxinpolyclonal antiserum were diluted 10-fold in PBS-T containing 0.2% BSAand exposed to 10 changes of E. coli lysate-treated nitrocellulosemembranes for 30 minutes duration each at 37° C. The resultantpolyclonal antisera had minimal reactivity against E. coli proteins.Neutralizing activity of the serum, as determined by the MTT dyeneutralization test and the indirect ELISA titer, were measured asdescribed previously (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa. 1992.Factors affecting leukotoxin activity of Fusobacterium necrophorum. Vet.Microbiol. 33:15-28; Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, and J.S. Smith. 1994. Biological and biochemical characterization ofFusobacterium necrophorum leukotoxin. Am. J. Vet. Res. 55:515-519; Tan,Z. L., T. G. Nagaraja, M. M. Chengappa, J. J. Staats. 1994. Purificationand quantification of Fusobacterium necrophorum leukotoxin usingmonoclonal antibodies. Vet. Microbiol. 42:121-133).

Extraction of Genomic Dna from F. Necrophorum and E. Coli

Chromosomal DNA was extracted from highly virulent F. necrophorum subsp.necrophorum, strain A25 (18) and E. coli DH5α. (F⁻ λ⁻ φ80 Δ[lacZYA-argF] endA1 recA1 hsdR17deoR thi-1 supE44 gyrA96 relA1), using amodification of the method described by Hull and coworkers (Hull, R. A.,R. E. Gill, P. Hsu, B. H. Minshew, and S. Falkow. 1981. Construction andexpression of recombinant plasmids encoding type 1 orD-mannose-resistant pili from a urinary tract infection Escherichia coliisolate. Infect. Immun. 33:933-938). E. coli was cultured in Luria brothwith shaking under aerobic conditions at 37° C. and F. necrophorum wasgrown overnight in a prereduced anaerobically sterilized brain heartinfusion broth in serum bottles under anaerobic conditions at 39° C.Cell pellets were resuspended in TES buffer (25% sucrose, 50 mM Tris-HCl[pH 7.5] and 1 mM EDTA); spheroplasted with lysozyme at room temperaturefor 30 min; and lysed using sarkosyl in the presence of proteinase K at60° C. for 1 hour. The product was extracted with buffer-saturatedphenol and chloroform, and the DNA was precipitated in 2.5 volumes ofice-cold ethanol. The DNA pellet was resuspended in TE buffer (10 mMTris-HCl [pH 8.0] and 1 mM EDTA) and subjected to ultra centrifugationin a cesium-chloride step-gradient (43.5% to 60%) containing ethidiumbromide (0.4 mg/ml final volume). The chromosomal DNA band was extractedwith TE buffer and CsCl saturated isopropanol to remove ethidium bromideand dialyzed against double-distilled water. The DNA concentration andpurity were checked spectrophotometrically.

Genomic Library and Screening

Genomic DNA of F. necrophorum A25 was digested partially withrestriction endonuclease Sau3AI, and the fragments weresize-fractionated in a sucrose gradient. Ten to 12 kb fragments werecloned into BamHI digested and alkaline phosphatase-treated Lambda zapExpress vector (Stratagene Corp. La Jolla, Calif.) as per themanufacturer's instructions. Recombinant lambda DNA was packaged(Gigapack gold; Stratagene) and used to infect XL1Blue MRF′ host cells(Stratagene). Plaques were lifted onto nitrocelluose membrane andscreened with antileukotoxin polyclonal antiserum using a Picoblueimmuno-screening kit as per the manufacturer's protocol (Stratagene).Immunoreactive clones were plaque purified three times using thepolyclonal antiserum. The recombinant DNA from immunoreactive clones wasrescued as phagemid (pBKCMV) clones using Exassist helper phage in E.coli XLOLR strain as per the manufacturer's protocol (Stratagene).

DNA Sequencing Analysis

Phagemids from immunoreactive clones, purified PCR products, and plasmidsubclones were sequenced using vector-specific or internal primers witha model 373A automated DNA sequencer (Applied Biosystems, Foster City,Calif). The DNA sequences were aligned and analyzed using Sequencher(version 3.1.1, Gene Codes Corp., Ann Arbor, Mich.) and DNA Strider(Version 1.2).

Inverse Per and Sequence Extension

Chromosomal DNA from F. necrophorum strain A25 was digested singly withrestriction endonucleases TaqI, EcoRI, DdeI, or Sau3AI. After completedigestion of the chromosomal DNA with any one of these enzymes, theproducts were extracted with phenol and chloroform, and precipitatedwith ethanol. Under dilute conditions (200 ng of digested DNA in 100 μmltotal volume), DNA was self-ligated using T4 DNA ligase at 16° C.overnight. Ligated DNA was extracted with phenol and chloroform,precipitated with ethanol and reconstituted in 10 ml of nuclease freewater. Two microliters of the ligated DNA were used as templates for 100ml PCR reactions with forward and reverse primers designed based on thesequence obtained from previous sequencing reactions. The products frominverse PCR were cloned in pCR TOPO cloning vectors (TA, Blunt2 orBlunt4) as per the manufacturer's instructions (Invitrogen Corp. SanDiego, Calif.), and sequenced directly or after subcloning, using vectorspecific primers. Six successive inverse PCRs were carried out to reachthe 3′ end of the leukotoxin gene.

Creation of Gene Truncations

Polymerase chain reaction using thermostable polymerase (EXTaq; TakaraCorporation, Madison, Wis.) was used to amplify five overlapping regionsof the leukotoxin gene ranging in size from 1.1 kb to 2.8 kb.Chromosomal DNA from F. necrophorum strain A25 was used as the template.The forward primers were designed to contain a SacI site, and thereverse primers had an XmaI site, for in-frame insertion into theHis-tag expression vector pQE30 (Qiagen Inc. Valencia, Calif.). Eachtruncated gene product overlapped with the adjacent product by at least100 bp. One kb of DNA from the 3′ end of the upstream open reading frame(ups) was amplified and cloned in pQE30 vector as described above.Recombinant plasmids were transformed into E. coli host strain M15 forinducible expression of proteins encoded by cloned genes under thecontrol of the lac promoter. The five truncated leukotoxin polypeptidesand the C-terminus of the upstream polypeptide were purified usingnickel chelation chromatography under denaturing conditions to apparenthomogeneity as indicated by silver-stained SDS-PAGE gels (data notshown).

Preparation of Polyclonal Antiserum Against the Truncated LeukotoxinPolypeptides

New-Zealand White rabbits were injected intramuscularly with the fivetruncated leukotoxin polypeptides or the upstream polypeptide (0.5mg/animal) precipitated with aluminum hydroxide. A booster dose wasgiven on day 21 (0.5 mg /animal). Serum samples were collected on days21 and 42 and antileukotoxin titers were determined by indirect ELISAusing affinity purified native leukotoxin (Tan, Z. L., T. G. Nagaraja,M. M. Chengappa, J. J. Staats. 1994. Purification and quantification ofFusobacterium necrophorum leukotoxin using monoclonal antibodies. Vet.Microbiol. 42:121-133.). Leukotoxin neutralizing activities of the 42day serum samples were determined by the MTT dye neutralization assayusing 200 units of toxin (id.).

Immunoblot Analysis

Affinity-purified native leukotoxin, the truncated leukotoxinpolypeptides and upstream polypeptide purified over nickel columns,whole cell lysates from bacterial clones carrying recombinant expressionplasmids, and concentrated culture supernatants were resolved bySDS-PAGE (6 or 10% acrylamide) and electroblotted to nitrocellulosemembranes (BioRad minigel II electrophoresis and transfer unit).Monoclonal antibody against native leukotoxin (F7B10) or polyclonalantisera raised against native leukotoxin, various truncated leukotoxinor upstream polypeptides were used to probe the western blottedproteins. Goat antimouse or antirabbit IgG conjugated to alkalinephosphatase (Sigma Chemical Company, St. Louis, Mo.) was used as thesecondary antibody, and the immunoreactive proteins were detected usingnitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate assubstrates.

Cloning and Expression of Full-length Leukotoxin ORF

A 4.3 kb DNA fragment containing the 5′ end of the lktA open readingframe up to the internal NheI restriction endonuclease recognition sitewas amplified from A25 chromosomal DNA. This fragment was cloned intothe kanamycin resistance encoding vector pCR Blut II TOPO. A 5.4 kb DNAfragment extending from the NheI site to the 3′ end of the lktA openreading frame was PCR amplified and cloned into the low-copy,spectinomycin resistance plasmid pCL1921 (Lerner, C. G., and M. Inouye.1990. Low copy number plasmids for regulated low level expression ofcloned genes in Escherichia coli with blue/white insert screeningcapability. Nucl. Acid. Res. 18:4631-4633.). The two resulting plasmidclones were ligated together making use of the unique NheI site presentin lktA ORF, and the transformants were selected on media containingspectinomycin (100 μg/ml) and kanamycin (21 μg/ml). The pCR Blunt Itvector specific sequences were then removed by digesting the resultantplasmid with SacI followed by ligation under dilute conditions andselection on L-agar containing 100 μg/ml spectinomycin. Thus the entire9,726 base pairs of the leukotoxin ORF were cloned in a low-copy numberplasmid pCL1921 to produce pSN1999. Making use of the unique XmaI siteintroduced into at the 3′ end of the open reading frame and the SacIsite introduced into the 5′ end of the reading frame, the entire lktAcoding sequence was cloned in-frame into the expression plasmid pQE30 togive pSN2000.

Flow Cytometric Analysis of Leukotoxin Biological Activity

Bovine peripheral polymorphonuclear leukocytes were isolated asdescribed previously (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa. 1992.Factors affecting leukotoxin activity of Fusobacterium necrophorum. Vet.Microbiol. 33:15-28; Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, and J.S. Smith. 1994. Biological and biochemical characterization ofFusobacterium necrophorum leukotoxin. Am. J. Vet. Res. 55:515-519).Untreated cells (negative control) or those treated with either 200units of native leukotoxin from F. necrophorum (positive control) orwhole-cell lysates from clones expressing full-length recombinantleukotoxin were tested for viability by flow cytometry (Facstar, BectonDickinson Immunocytometry Systems, San Jose, Calif.). Briefly, 1 ml ofbovine peripheral PMNs (9×10⁶ cells/ml) was incubated with variouspreparations of toxin for 45 min at 37° C. in a chamber containing 5%CO₂. The cells were then washed twice in 2 ml of HBSS (pH 7.2) andresuspended in 300 μl of HBSS. These cells were treated for 10 min inthe dark at room temperature with 10 μl of 5 mg/ml propidium iodide(PI). The red fluorescence (FL-2 [585/42]) is proportional to the numberof cells which have lost membrane integrity and, therefore, do notexclude the propidium iodide. Leukocyte subpopulations were displayed ina dot plot and gated according to size based on forward scatter (FSC)and granularity or 90 degree light scatter (SSC). A region was placedaround granulocytes, cells of larger size and granularity and thusexcluding monocytes, and data were collected on 10,000 gated cells. Theidentity of the gated cells as granulocytes by was indicated by indirectimmunofluorescence labelling with monoclonal antibody DH59B (VMRD Inc.,Pullman, Wash.) which reacts with the granulocyte-monocyte-1 receptor.Fluorescence signals displayed as a dot plot were used to determine thepercent positive cells by quadrant statistics.

Southern Blot Analysis

Genomic DNA was extracted from several strains of F. necrophorum subsp.necrophorum and subsp. funduliforme isolated from ruminal contents orliver abscesses. Chromosomal DNA was digested to completion with HaeIII,which cleaves the leukotoxin ORF once. The digested DNA waselectrophoresed in a 1% agarose gel and Southern blotted onto anitrocellulose membrane. The full-length lktA ORF cloned in pQE30(pSN2000) was released by digestion with SacI and XmaI, and the insertDNA was gel purified, radiolabelled with [α-³⁵S]dATP, and hybridized.

Nucleotide Sequence Accession Number

The nucleotide sequence of F. necrophorum subsp. necrophorum strain A25lktA has been assigned GenBank accession number AF312861.

Cloning and Nucleotide Sequence of the F. Necrophorum LeukotoxinDeterminant

A Sau3A-generated genomic library of F. necrophorum strain A25 DNA wasscreened using rabbit polyclonal antisera raised againstimmunoaffinity-purified native leukotoxin and immunoreactive clones wereidentified. The clones carried inserts of approximately 4.6, 5.5, and6.3 kb in length. The immunoreactive clones containing the leukotoxinopen reading frame (designated lktA) are depicted in FIG. 1. Inverse PCRwas used to extend the cloned region to allow completion of the sequenceof the lktA open reading frame. The 11, 130 bp sequence of F.necrophorum DNA contained one complete and two partial ORFs. Theupstream (orfB) partial ORF comprises the first 1,018 bp. The lktA ORFinitiates 16 bp downstream of the lktB ochre codon. A putativeribosome-binding site (RBS) with the sequence AAGGGGGT precedes the lktAORF. The first two bases of the RBS were the last two bases of the lktBstop codon. The leukotoxin determinant is 9,726 bp and encodes a proteinof 3,241 amino acids with an overall molecular weight of 335,956. Thededuced protein sequence is unusual in that it lacks cysteine residues.The protein has substantial hydrophobic character (FIG. 5) and possesses14 regions with sufficient hydrophobic character and length to bemembrane spanning. However, this is a secreted toxin in F. necrophorum.The potential transmembrane domains may provide a clue as to the mode ofaction of the leukotoxin on the target neutrophils.

A BLAST search of the protein database with the deduced leukotoxin didnot indicate significant sequence similarity to any bacterialcytotoxins. Some sequence similarity, generally 17-20% amino acididentity over a window of 1,500 to 2,000 residues, was found to certainhigh molecular weight cell surface proteins. These include the SrpAserine-rich protein from Streptococcus cristatus (accession numberU96166), the hemagglutinin from Streptococcus gordonii (AB029393), asurface protein from Xylella fastidiosa (AE003982), the outer membraneprotein A from Rickettsia australis (AF149108), the 190 kDa surfaceantigen precursor from R. rickettsii (A41477), and the high molecularweight antigen (HmwA) of Haemophilus influenzae (AF180944). Given themolecular size of the leukotoxin protein, which is larger than any knownbacterial exotoxin, its lack of cysteine residues, and its lack ofsequence similarity to other bacterial leukotoxins, the LktA proteinfrom F. necrophorum appears to be a novel leukotoxin.

The deduced amino acid sequence of the carboxy terminus of the OrfBprotein has some sequence identity to heme-hemopexin utilization protein(HxuB) of Haemophilus influenzae (21% amino acid identity over a 283residue window). The putative open reading frame upstream of theleukotoxin determinant does encode a protein product. The 1 kb sequenceencoding the carboxyl terminus of this ORF was cloned into pQE30, andthe polypeptide was expressed with the six histidine tag at itsN-terminus. The protein was purified by nickel chelation chromatography,and the antiserum was raised against this protein in rabbits. Westernblot analysis revealed that this antiserum recognized a 60 kDa proteinin whole-cell lysates of F. necrophorum (data not shown). This proteinwas not present in culture supernatants or in purified outer membranesof F. necrophorum.

Downstream of lktA is another apparent open reading frame, which extendsto the end of the cloned sequences (375 bp). The putative ATG startcodon overlaps the opal stop codon of lktA. The nucleotide and deducedamino acid sequences do not show significant sequence similarity to anysequences currently in GenBank.

Creation of Truncated Leukotoxin Polypeptides and Characteristics ofPolyclonal Antisera Raised Against them

A 3.5 kb sequence from the 5′ end of lktA gene was amplified by PCR andcloned in-frame in the expression vector pQE30. Induced expression ofthis truncated version of the leukotoxin protein with IPTG resulted inthe immediate cessation of growth and lysis of the host E. coli cells.In order to obtain better expression of recombinant protein and lesstoxicity to E. coli host cells, smaller truncations of the leukotoxingene were constructed. The truncated polypeptides were named BSBSE, SX,GAS, SH, and FINAL starting from the N-terminus and ending at theC-terminus of the leukotoxin protein (FIG. 6). In this Figure, the boxesrepresent the leukotoxin open reading frame (lktA) and its flankingputative open reading frames. The lines above the boxes represent thephagemid clones (816, 101, and 611) obtained from the immunoreactiveplaques in the cloning experiments. The region designated iPCRrepresents the sequence obtained from sequencing a series of inverse PCRclones. The plasmid pSN2000 contains the entire lktA open reading frame.Below the boxes are the clones expressing the truncated leukotoxinpolypeptides. The numbers refer to the nucleotide positions of theboundaries of each truncation relative to the 11,130 bp sequencedeposited in GenBank.

Each polypeptide had an overlap of at least 21 amino acids with itsadjacent polypeptide. The C-terminal truncated polypeptide of theupstream protein and the polyclonal antiserum raised against it, servedas a negative control in our toxicity and toxin-neutralization studies.Purified truncated leukotoxin and upstream polypeptides were thenanalyzed by western blots, for their reactivity against polyclonal andmonoclonal antisera raised against affinity-purified native leukotoxin,using western blot analysis. Antileukotoxin polyclonal antisera reactedstrongly with polypeptides BSBSE, SX, and FINAL and weakly withpolypeptides GAS and SH (FIG. 7a). Monoclonal antileukotoxin antibodyreacted with the N-terminal polypeptide, BSBSE, but not any othertruncated leukotoxin polypeptides (FIG. 7b). As expected, the UPSpolypeptide did not react with polyclonal or monoclonal antileukotoxinantibodies. Polyclonal antisera raised in rabbits against each of thetruncated leukotoxin polypeptides reacted strongly with thecorresponding polypeptide and also the native leukotoxin. These resultsare shown below in Table 1. Antibodies raised against individualtruncations reacted weakly to their adjacent polypeptides because of thepresence of the overlapping amino acid sequences between them (data notshown). Antiserum raised against UPS (from the upstream ORF) failed torecognize the leukotoxin.

TABLE 1 Neutralization of Leukotoxin from F. Necrophorum by RabbitPolyclonal Antisera Raised Against the Recombinant TruncatedPolypeptides. ELISA Titer Neutralization Immunogen Self polypeptideNative Leukotoxin Titer UPS 9,600 ± 1,693 19 ± 17 <5 BSBSE 10,420 ±1,142  10,680 ± 1,653  1,460 ± 71 SX 8,754 ± 983   7,480 ± 1,593 <5 GAS8,748 ± 865   8,100 ± 1,297 1,280 ± 89 SH 10,180 ± 1,789  8,220 ± 1,301<5 FINAL 9,750 ± 1,343 9,440 ± 1,262 <5 ELISA titers are presented asthe mean of three determinations expressed as the reciprocal of thehighest dilution giving a positive reaction (± standard deviation). Theneutralization titer is the reciprocal of the greatest dilution ofantiserum able to neutralize the activity of 200 units of nativeleukotoxin in an MTT assay.

Antisera raised against the individual polypeptides were tested forneutralization activity against the native leukotoxin from F.necrophorum. An ELISA assay was utilized to measure the reactivity ofeach antiserum against the leukotoxin. An MTT dye reduction assay wasthen utilized to determine if the antiserum could neutralize the toxiceffects of the leukotoxin against bovine peripheral leukocytes. As shownin Table 1, two of the antisera could neutralize the leukotoxin. Theactive antisera were raised against the N terminal polypeptide (BSBSE)and the middle polypeptide (GAS). The other three antisera did not haveneutralizing activity in this assay, although the ELISA data indicatedthat each antiserum recognized the F. necrophorum leukotoxin.

Creation of Full-length Recombinant Leukotoxin and its Toxicity toBovine Peripheral Blood Polymorphonuclear Cells

The entire leukotoxin gene (9,726 bp)was cloned into the pQE30expression vector. Unlike certain truncated versions of the leukotoxinprotein, full-length recombinant leukotoxin upon expression was nottoxic to E. coli host cells. When whole-cell lysates from clonesexpressing full-length leukotoxin were subjected to western blot assays,both polyclonal (not shown) and monoclonal antileukotoxin antibodiesreacted to high-molecular weight (>220 kDa) protein species (FIG. 7c).In this Figure, MW is molecular weight markers; Lkt, isaffinity-purified leukotoxin from F. necrophorum; FL-I and FL-UI arefull-length clone induced or uninduced with IPTG; Super is concentratedF. necrophorum A25 culture supernatant. Additionally, the arrows denotethe positions of the reactive BSBSE band in FIG. 7b and the full-lengthleukotoxin in FIG. 7c. The amount of full-length leukotoxin in theculture supernatant in panel C was insufficient to be visualized as adistinct band in this blot. The protein was extremely unstable, asevident by the presence of numerous smaller molecular weight species,which presumably represent breakdown products. This instability was alsoobserved with native leukotoxin that was immunoaffinity-purified from F.necrophorum culture supernatants. Antisera raised against all thetruncated leukotoxin polypeptides, including the C-terminal FINALpolypeptide, reacted to recombinant leukotoxin suggesting that theprotein may be expressed in its full-length (data not shown). Asexpected, antibody raised against the upstream polypeptide failed toreact to the full-length recombinant leukotoxin.

Bovine peripheral polymorphonuclear leukocytes exposed to whole-celllysates of full-length or truncated recombinant clones (12 mg/mlprotein) prior to or after induction with IPTG were tested for membraneintegrity using propidium iodide exclusion and flow cytometry. Controlcells untreated with leukotoxin gave a baseline value of 5.4%PI-staining cells (FIG. 8). In this Figure, membrane damage was assessedby staining of the cells with propidium iodide. Shown are the valuesobtained after counting 10,000 PMNs (stippled bars) or the lymphocytefraction (hatched bars). Cells were untreated (control), treated with200 units of affinity purified leukotoxin from F. necrophorum (Fnleukotoxin) or lysates of E. coli harboring expression plasmids bearingthe upstream polypeptide (pSN100) or the full-length lktA open readingframe (pSN2000). The “U” and “I” designations refer to lysates fromuninduced cultures and cultures induced with 1 mM IPTG, respectively.Induced lysates were also tested after 1:5, 1:25, and 1:125 dilutions inPBS. The results shown are the averages of three experiments and thestandard deviation is indicated.

The addition of 200 MTT units of affinity-purified native leukotoxinresulted in 75.4% of the PMNs taking up the dye. An MTT unit of thetoxin is defined as the reciprocal of the dilution causing a 10%decrease in MTT-dye reduction activity. The affinity-purified leukotoxinpreparation used in this study had an activity of 2×10⁵ units/ml.Lysates from the clone expressing the upstream polypeptide (SN100) didnot increase the percentage of PI-staining cells, indicating that thetruncated form of this protein lacked membrane-damaging activity.Whole-cell lysates from E. coli carrying recombinant full-lengthleukotoxin gene (SN2000), uninduced with IPTG, gave rise to 9.6%PI-staining bovine PMNs, whereas lysates from induced clones gave 27.3%staining PMNs. The low percentage of damaged cells from the uninducedlysate resulted from leaky expression of the toxin with this vector,consistent with the results obtained by western blot analysis (notshown). The membrane damaging activity in the induced lysate wasproportionately lost when the samples were diluted in phosphate-bufferedsaline. The data indicate that recombinant full-length leukotoxin istoxic to bovine neutrophils.

Preparations of PMNs had residual contaminating cells of smaller sizeand granularity, which were found to be predominantly lymphocytes byimmunophenotyping with anti-CD3 and anti-IgM specific monoclonalantibody. These cells were gated, and the effects of various leukotoxinpreparations on the viability of these cells were measured as describedfor PMNs. Untreated control lymphocytes gave a baseline value of 13.6%staining cells, whereas inclusion of 200 units of affinity-purifiednative leukotoxin resulted in 31.3% of the lymphocytes taking up the PI(FIG. 8). The apparently lower sensitivity of lymphocytes compared toPMNs is characteristic of F. necrophorum leukotoxin. Furthermore, therecombinant toxin displayed the same degree of activity againstlymphocytes as did the native leukotoxin. Among lymphocytes treated withlysates from E. coli carrying uninduced recombinant full-length lktA,12.8% were PI-positive compared to 19.2% obtained with lysates frominduced clones. Thus the expressed recombinant leukotoxin hadtoxicological properties similar to those of the native leukotoxinpurified from F. necrophorum culture supernatant. Lysates from E. coliwith IPTG-induced expression of the leukotoxin truncated polypeptides orthe upstream polypeptide did not display membrane-damaging activityagainst either bovine PMNs or the lymphocyte-containing population (FIG.9). In this Figure, membrane damage was assessed by staining of thecells with propidium iodide. Shown are the values obtained aftercounting 10,000 PMNs (stippled bars) or the lymphocyte fraction (hatchedbars). Cells were untreated (control), treated with 200 units ofaffinity purified leukotoxin from F. necrophorum (native toxin), lysatesfrom IPTG-induced cultures of clones expressing the truncatedpolypeptides (ups, BSBSE, SX, GAS, SH, and Final) or the wholerecombinant leukotoxin (whole toxin). The results shown are the averagesof three experiments and the standard deviation is indicated.

Presence of the Leukotoxin Determinant in F. Necrophorum Isolates

The leukotoxin gene was cloned and sequenced from F. necrophorum subsp.necrophorum A25, a strain originally isolated from a bovine liverabscess. Southern blot hybridization of the chromosomal DNA extractedfrom various F. necrophorum strains of both subspecies isolated fromruminal contents or liver abscesses was carried out using the leukotoxinORF as a probe (FIG. 10). In this Figure, F. necrophorum subsp.necrophorum from liver abscesses are in lane 1 which is strain A21; lane2 which is A25; and lane 3 which is A39. F. necrophorum subsp.necrophorum from ruminal contents are in lane 7 which is RA13; lane 8which is RA15; lane 9 which is RA16; lane 10 which is RA18; lane 11which is RA26; lane 12 which is RA28; and lane 13 which is RA29. The F.necrophorum subsp. funduliforme isolates from liver abscesses are inlane 4 which is B17; lane 5 which is B29; lane 6 which is B35 or ruminalcontents which are in lane 14 which is RB33; and lane 15 which is RB37.Strains are described in reference 24. M, DNA molecular weight markers.The restriction endonuclease HaeIII was used to digest the chromosomalDNA from F. necrophorum isolates. A single recognition site for thisenzyme occurs 5,933 bp from the start codon in the lktA ORF. Thus, twohybridizing fragments should be present in strains carrying this gene.All strains of F. necrophorum subsp. funduliforme isolated from liverabscesses (B17, B29, and B35) or ruminal contents (RB33 and RB37) wereidentical in their hybridization patterns showing two bands atapproximately 7 and 8 kb each. Also, all isolates of F. necrophorumsubsp. necrophorum, except A39, isolated from liver abscesses (A21 andA25) and those isolated from ruminal contents (RA13, RA15, RA16, RA18,RA26, RA28, and RA29) had identical hybridization patterns showing twobands of approximately 10 and 11 kb each. A single band of approximately10.5 kb, presumably a doublet, hybridized to the leukotoxin gene inchromosomal DNA of strain A39 (FIG. 10, lane 4). This suggests that someheterogeneity may be present in the leukotoxin locus sequences amongstrains of F. necrophorum subsp. necrophorum. However, the hybridizationpattern does appear to be a good indicator for subspecies determination.

EXAMPLE 3 Construction of Truncated Forms of the Leukotoxin

A 3.5 kb sequence from the 5′ end of lktA gene was amplified by PCR andcloned in-frame in the expression vector pQE 30 (Qiagen Corporation).Induced expression of this truncated version of the leukotoxin proteinwith IPTG resulted in the immediate cessation of growth and caused lysisof the host E. coli cells. In order to obtain better expression ofrecombinant protein, smaller truncations of the leukotoxin gene wereconstructed. Polymerase chain reaction using thermostable polymerasewith proofreading ability (EXTaq; Takara Corp.) was used to amplify fiveoverlapping regions of the leukotoxin gene. The forward primers weredesigned to contain a SacI site, and the reverse primers had a XmaIsite. F. necrophorum A25 chromosomal DNA was used as the template, andthe amplified products were digested with restriction enzymes SacI andXmaI, and cloned in-frame in the His-tag expression vector pQE 30. Fivetruncated leukotoxin proteins and the C-terminus of the upstream proteinwere purified using nickel chelation chromatography to apparenthomogeneity as indicated by silver-stained SDS-PAGE gels. The proteinswere then tested for their reactivity with polyclonal antisera raised inrabbits against affinity purified native leukotoxin using western blotanalysis. Purified proteins were injected in rabbits to producepolyclonal antisera, which in turn were used to carry out western blotanalysis and neutralization tests (Table 2). Antisera raised againsteach protein recognized native leukotoxin from F. necrophorum. Antiseradirected against the BSBSE9 and GAS polypeptides were able to neutralizethe activity of native leukotoxin. Thus the cloned ORF does indeedrepresent the F. necrophorum leukotoxin.

TABLE 2 Characterization of the Truncated Upstream and LeukotoxinProteins Antisera Antisera Raised Neutral- Truncated Against izes Leuko-Recognized Truncated Activity of toxin Number by Proteins Leuko-Proteins of Anti-native Recognized toxin (N to C Amino Size (inLeukotoxin Native Against terminal) Acids Daltons) Antibodies LeukotoxinPMNs UPS 9 339 38324 − − − BSBSE 9 377 40810 + + + SX7 926 97453 + + −GAS 15 713 71949 + + + SH 12 628 63457 + + − FINAL 2 774 80590 + + −

Production of an Inactivated Recombinant Leukotoxin Vaccine

The immunogenicity and protective immunity of the recombinant fulllength and truncated leukotoxin proteins is determined in mice andcompared to the efficacy of immunization with inactivated nativeleukotoxin in F. necrophorum culture supernatant. The usefulness of themouse model in studying experimental Fusobacterium infections has beenwell documented (Abe et al., 1986, Emery and Vaughn, 1986).

Vaccine Preparations

Purified recombinant leukotoxins (described above) including thefull-length protein are inactivated by the addition of formalin (finalconcentration 0.3%) and homogenized with Ribi or other suitable adjuvant(10% vol/vol; Ribi Immunochem, Hamilton, Mont.). The native leukotoxoidvaccine is prepared with culture supernatant from F. necrophorum subsp.necrophorum, strain A25 grown in PRAS-BHI broth (Saginala et al., 1997).The leukotoxic activities of the recombinant leukotoxin and culturesupernatant, before and after formalin inactivation, are then tested byMTT-dye reduction assay using bovine polymorphonuclear (PMN) leukocytesas target cells (Tan et al., 1992). The quantity of native leukotoxin isthen assayed using a sandwich ELISA using purified monoclonal antibody(Tan et al., 1994b).

Immunogenicity of the Inactivated Recombinant Leukotoxin in Mice

Immunogenicity and protective effects of the inactivated recombinantfull length, and truncated leukotoxins are evaluated in comparison withthe native leukotoxin (culture supernatant of F. necrophorum, strainA25). Five overlapping truncations and the recombinant full-lengthleukotoxin are purified using the nickel-affinity columns. The treatmentgroups include control (0.2 ml PBS), native leukotoxin, recombinant fulllength, and truncated leukotoxins individually or in combination (allfive truncations individually, and a mixture of all five truncatedproteins in equimolar ratio). Additionally, a mixture of the twotruncated proteins BSBSE and GAS in equimolar concentrations is testedfor immunogenicity, because polyclonal antisera raised against these twoproteins neutralize the activity of native leukotoxin against bovineneutrophils. Each leukotoxin preparation is tested at 10 and 50 μg doses(total protein concentration), administered subcutaneously on days 0 and21. Six mice (7-8 wk old BALB/c) are used in each treatment group. Bloodsamples are collected on days 0, 14, 21, 35, and 42. Serum is stored at−70 C. until assayed for antileukotoxin antibody. After the last bloodsampling (on day 42), mice are challenged intraperitoneally with 0.4 mlof late-log phase F. necrophorum strain A25 culture (6-7 hour culture inPRAS-BHI broth with an absorbance of 0.65 at 600 nm and with a cellconcentration of approximately 1 to 5×10⁸ CFU/ml). The number ofbacteria used for inoculation is enumerated by viable counts on bloodagar plates in an anaerobic glove Box (Forma Scientific, Marietta,Ohio). Mice are observed for 4 days after challenge to record mortalityand clinical signs, and those that survive the challenge are euthanized.Mice are then necropsied and examined grossly for abscesses in theliver. Additionally, other organs and liver tissue will be cultured foranaerobic bacterial isolation.

Following this study, the efficacious dose and the recombinantleukotoxin preparation is selected and one more immunization andchallenge study in mice to confirm the protective effect of recombinantleukotoxin is conducted. Groups of 7-8 week old BALB/c mice (10 pergroup) are used and each group receives one of the following leukotoxinpreparations: most immunogenic recombinant leukotoxin protein,combination (two or more) of most immunogenic recombinant leukotoxinproteins, and native leukotoxin (F. necrophorum culture supernatant).The leukotoxin proteins are inactivated with 0.3% formalin, mixed withRibi or any other suitable adjuvant and emulsified with a homogenizerand administered subcutaneously on days 0 and 21. Blood samples arecollected on days 0, 14, 21, 35 and 42. Serum samples are assayed forantileukotoxin antibody. After the last blood sampling (on day 42), miceare challenged as described above. Overlapping variants of effectivepolypeptides (the truncated protein fragments) are identified and areconstructed in order to identify the polypeptide sequences that are mosteffective in conferring protection.

Determination of Antileukotoxin Antibody Induced by Immunization

Mouse serum is analyzed for antileukotoxin antibody by two methods.First, serum samples are assayed for leukotoxin neutralizing antibody bytesting its ability to neutralize the toxin using the MTT dye reductionassay with mouse and bovine PMNs as the target cells (Saginala, et al.,1996b; Tan et al., 1994a). Second, serum samples are tested foranti-leukotoxin IgG antibodies by enzyme linked immunosorbent assay(ELISA) using affinity-purified leukotoxin as the coating antigen.Affinity purification of the leukotoxin is carried out using monoclonalantibody MAbF7B10 (Tan et al., 1994b).

EXAMPLE 4 DNA Extraction and Polymerase Chain Reaction

Chromosomal DNA was isolated from F. necrophorum subspecies necrophorum,strain A25. Briefly, F. necrophorum was grown overnight in a PRAS-BHIbroth in serum bottles at 39° C. Cell pellets were resuspended in TESbuffer (25% sucrose, 50 mM Tris-HCl [pH 7.5] and 1 mM EDTA),spheroplasted with lysozyme at room temperature for 30 min, and lysedusing sarkosyl in the presence of proteinase K at 60° C. for 1 hour. TheDNA was extracted with buffer-saturated phenol and chloroform and wasprecipitated in 2.5 volumes of ice-cold ethanol and {fraction (1/10)}volume of sodium acetate (3 M, pH 5.2). The DNA pellet was resuspendedin TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) and was run for 20hours in a cesium-chloride gradient (60% to 43.5%) containing ethidiumbromide (0.4 mg/ml final volume). The chromosomal DNA band was extractedwith cesium-chloride saturated isopropanol to remove ethidium bromideand dialyzed against double distilled water. DNA concentration andpurity were checked spectrophotometrically.

The primers were designed to amplify the leukotoxin gene as fiveoverlapping truncations (Table 3). The sites for annealing of theprimers were chosen, so that there is an overlap of approximately 100 bpwith the adjacent truncated leukotoxin gene product. Each forward primerwas designed to contain a SacI site and reverse primers carried a XmaIsite (Table 3). PCR amplifications were carried out under followingconditions using a thermostable DNA polymerase with a proof-readingfunction ExTaq (Takara Corp., Madison, Wis.): initial denaturation 94°C. for 3 min; 36 cycles of denaturation 94° C. for 1 min, 59° C. for 45sec, 67° C. for 30 sec, and 72° C. for 1 to 3 min (at min per kb), and afinal extension at 72° C. for 4 min.

TABLE 3 PCR primers used for amplifying truncated leukotoxin genesegments. Truncated Location in segment lktA gene (bp) DesignationPrimer Sequence^(a) bsbse  1-22 BS-START tccgagctcATGAGCGGCATCAAAAATAACG1130-1112 BS-END tcgccccgggATAGGAGAAATAGAACCTG sx 919-940 SX-STARTtccgagctcGGGAGATTTATAAAGAAAGAAG 3698-3679 SX-ENDtcgccccgggGATCCGCCCCATGCTCCAAC gas 3553-3572 GAS-STARTtccgagctcGGAGCTTCTGGAAGTGTTTC 5693-5674 GAS-ENDtcgccccgggGTACTATTTTTTATATGTGC sh 5623-5641 SH-STARTtccgagctcGCTGCAGTAGGAGCTGGAG 7510-7492 SH-ENDtcgccccgggCTGCAGTTCCCAAACCACC final 7405-7425 FIN-STARTtccgagctcGGAATTAAAGCCATTGTGAAG 9726-9706 FIN-ENDtcgccccgggTCATTTTTTCCCTTTTTCTCC ^(a)Lower case letters in primersequences represent extra bases added to incorporate restriction sites.

Directional Cloning in an Expression Vector

The amplified gene products which are overlapping truncations extendingfrom 5′ to 3′ end of the leukotoxin gene (lktA), were named BSBSE, SX,GAS, SH, and FINAL (FIG. 11). In this Figure the numbers in parenthesesindicate the size in kilobases of each insert. They were extracted withphenol and chloroform and precipitated with ethanol as described above.The amplified lktA gene products and expression vector pQE30 (QiagenCorp., Valencia, Calif.) were digested with restriction endonucleasesSacI and XmaI as per manufacturer's instructions (New England Biolabs,Beverly, Mass.). After digestion, the vector and insert DNA were phenoland chloroform extracted, ethanol precipitated, and ligated overnight at16° C. using T4 DNA ligase (Promega Corp., Madison, Wis.). Ligated DNAwas digested with restriction enzyme KpnI before transforming chemicallycompetent E. coli M15 cells as per standard procedures. Restrictionsites for KpnI is absent in the entire lktA gene and present in a singlelocation between SacI and XmaI sites in pQE 30. The expression vectorpQE 30 lacks blue/white selection, thus the above manipulation helped usto enrich clones that carry truncated leukotoxin gene products. Thetransformants were plated on Luria-agar plates containing ampicillin(100 ug/ml) and kanamycin (20 ug/ml) to select for clones containingplasmids pQE 30 and pRep4.

Expression of Truncated Leukotoxin Polypeptides

Plasmid DNA from the transformants were purified using Wizard SVminiprep columns (Promega), and the orientation of the insert waschecked by sequencing with a vector specific 5′QE primer which annealsupstream to the MCS using a Applied Biosystems 373A automated sequencer.Positive clones were induced for the expression of polypeptides withIPTG, the whole cell lysates from uninduced and induced were comparedfor immunoreactive polypeptides in a western-blot using polyclonalantisera raised in rabbits against affinity purified native leukotoxin(Tan et al, 1994d).

Antigen Preparation

Due to the presence of its codons in the sequence upstream of the MCS inthe vector pQE 30, six histidine residues are added in the N-terminus ofthe expressed polypeptides. The expressed polypeptides were purifiedusing nickel-affinity columns under denaturing conditions usingguanidium hydrochloride, as per the manufacturer's instructions(Qiagen). The column purified polypeptides were dialyzed for 48 hours at4° C. against sterile phosphate buffered saline (0.1 M, pH 7.2) toremove any traces of urea, and concentrated in Ultrafree-Biomax 30filters (Millipore Corp. Bedford, Mass.), which retains molecules ofsizes over 30 kDa. The protein concentrations were analyzed using theBCA assay (Pierce, Rockfort, Ill.) and the purity checked with SDS-PAGEanalysis followed by silver staining. Native leukotoxin from F.necrophorum culture supernatant was purified using immunoaffinitycolumns with anti-leukotoxin monoclonal antibody (F7B10) as describedpreviously. Also, leukotoxoid vaccine (12 hours culture supernatantinactivated with 0.3% formaldehyde) was made as described previously(Saginala et al., 1997).

Preparation of Polyclonal Antiserum Against Truncated Polypeptides

Five New-Zealand White rabbits were injected intramuscularly with thefive truncated leukotoxin polypeptides (0.5 mg/animal) precipitated withaluminum hydroxide. A booster dose was given on day 21 (0.5 mg/animal).Serum samples were collected on days 21 and 42 and antileukotoxin titerswere determined by indirect ELISA using affinity purified nativeleukotoxin. Leukotoxin neutralizing activities of the 42 day serumsamples were determined by the MTT dye neutralization assay. Aneutralization ratio, which was the dilution of the antiserum thatcaused neutralization divided by its ELISA titer, was calculated foreach truncated polypeptide.

EXAMPLE 5 Vaccine and Immunization

One hundred (100) 8 to 10 week old mice, identified by ear-markings,were randomly divided into 10 groups of 10 mice each. The groupsreceived five truncated leukotoxin polypeptides (BSBSE, SX, GAS, SH, andFINAL) individually, a mixture of BSBSE and GAS, a mixture of all fivetruncated polypeptides, affinity purified native leukotoxin, inactivatedculture supernatant, or PBS emulsified with Ribi adjuvant. Each mousewas injected subcutaneously (in two locations of 100 μl each between theshoulder blades) on day 0 and day 21 with 200 μl of one of the abovepreparations. The total amount of antigen in each injection (except withculture supernatant or PBS) was 10 μg per animal. Inactivated culturesupernatant (12 mg/ml protein concentration) was used without dilutionto reconstitute Ribi adjuvant (Ribi Immunochem, Hamilton, Mont.) andeach mouse was injected with 200 μl (2.4 mg protein) of the emulsifiedpreparation. Negative control group received 200 μl of PBS emulsifiedwith the Ribi adjuvant.

EXAMPLE 6 Determination of Antileukotoxin Antibodies Induced byImmunization

Blood for serum separation was collected from the right saphenous veinof each mouse on days 0, 21 and 42, and directly from the heart aftereuthanasia. Antileukotoxin antibody titers were assayed by an indirectELISA as described previously with slight modifications. Briefly,96-well microtiter plates (Falcon Probind assay plates, BecktonDickinson Labware, Lincoln Park, N.J.) were coated with 50 μl (2 μg/ml)per well of affinity purified native leukotoxin at 37° C. for 2 hours.The wells were blocked with 3% bovine serum albumin (Sigma ChemicalCompany, St. Louis, Mo.) in PBS at 37° C. for 2 hours. Fifty μl of a 1in 25 dilution of serum samples in PBS-T (0.05% Tween 20 in PBS) wereadded in duplicate and the plates were incubated at 37° C. for 1 hour.Following 6 washes with PBS-T, 100 μl of biotinylated goat anti-mouseimmunoglobulin (Accurate Chemicals and Scientific Corp., Westbury, N.Y.)was added to each well and incubated at 37° C. for 1 hour. The plateswere washed 6 times with PBS-T and 50 μl of streptavidin conjugated withhorseradish peroxidase was added to each well, and incubated at 37° C.for 1 hour. After washing the wells 6 times with PBS-T, 100 μ of ABTSsubstrate (2,2′-azino-di-[3-ethyl-benzthiazoline-6-sulfonic acid];Sigma) and H₂O₂in phosphate-citrate buffer (pH 4.0) was added to eachwell, and the plates were incubated for 30 min, or until colordevelopment, at room temperature. The absorbance was measuredcolorimetrically at 410 nm in a 96-well plate reader (Molecular Devices,Calif.).

EXAMPLE 7 Experimental Challenge with Fusobacterium necrophorum

Fusobacterium necrophorum subsp. necrophorum, strain A25 was grown to anOD₆₀₀ of 0.7 in PRAS-BHI broth and 0.4 ml of this late-log-phase culturewas injected intraperitoneally in mice. The inoculum had a bacterialconcentration of 4.7×10⁸ CFU/ml as determined by spread-plating on bloodagar plates Remel, Lenexa, Kans.) incubated in an anaerobic glove box(Forma Scientific, Marietta, Ohio). Mice were observed for 4 dayspost-challenge to record clinical signs and mortality. Mice thatsurvived for 4 days post-challenge were euthanized, necropsied andexamined for the presence of abscesses in liver and other internalorgans.

EXAMPLE 8 Enumeration of Fusobacterium necrophorum Load in the Liver

Livers from mice were collected at necropsy, weighed and homogenized ina tissue homogenizer for 1 min in PRAS-BHI broth. A 10-fold dilution ofthe homogenate was taken inside an anaerobic Glove box for furtherprocessing. Two hundred μl of modified lactate medium was dispensed intoeach well of the 96-well tissue culture plate (Falcon, Beckton DickinsonLabware, Lincoln Park, N.J.). Fifty μl of 1 in 10 dilution ofhomogenated liver was transferred to the wells on the first lane (8wells) and serially diluted (five-fold) up to the eleventh well. Thewells in the 12th lane were negative controls. The plates were incubatedin a Glove box at 39° C. for 48 hours. Kovac's reagent (20 μls each) wasadded to each well to detect indole production, presumptive of F.necrophorum. The bacterial load of F. necrophorum in liver wasenumerated by most probable number (MPN) analysis (Rowe, R., Todd, R.,and Waide, J. 1977. Microtechnique for most-probable-number analysis.Appl. Environ. Microbiol. 33:675-680.). Homogenized liver tissue sampleswere also streaked on blood agar plates and colonies identified usingRapid ANAII system (Innovative Diagnostic Systems, Norcross, Ga.).

EXAMPLE 9 Statistical Analyses

Serum ELISA measurements (absorbance values per ml of serum) wereanalyzed using Proc Mixed procedure of SAS (SAS systems, Cary, N.C.).The weights of liver and bacterial counts, log-transformed, wereanalyzed using PROC GLM program of SAS. P-values less than 0.01 wereconsidered significant.

Results Cloning and Expression of Leukotoxin Gene Truncations

In-frame cloning of the PCR amplified truncations of the leukotoxin gene(lktA) in plasmid pQE 30 was carried out as described above byincorporating restriction sites for SacI and XmaI in the forward andreverse primers respectively. Inducing the clones carrying varioustruncations did not produce inclusion bodies in the E. coli host cells.However, purification of the expressed polypeptides under nativeconditions was unsuccessful. Therefore, polypeptides were purified usingnickel affinity columns after denaturation with guanidiumisothiocyanate. The denatured truncated polypeptides, after dialysisagainst PBS, lacked toxicity to PMNs.

Antileukotoxin Antibody Titers in Rabbits

The anti-leukotoxin antibody titers in rabbits injected with truncatedpolypeptides are shown below in Table 4. Antisera raised againsttruncated leukotoxin polypeptides, BSBSE and GAS, neutralized thetoxicity of affinity purified native leukotoxin against bovineperipheral PMNs. The neutralizing activities for polyclonal antiseraraised against BSBSE and GAS were similar as evident from theiridentical neutralization ratios (0.146).

TABLE 4 Anti leukotoxin antibody titers in rabbits injected withtruncated leukotoxin proteins Neutral- Neutral- LISA LISA izationization Truncated Size (in titer on Titer on titer on ratio proteinsdaltons) day 21 day 42 (b) day 42 (a) (a/b) BSBSE 40810 1250 10000  14600.146 SX 97453 1000 8750 0 0 GAS 71949 1150 8750 1280 0.146 SH 634571000 10000  0 0 FINAL 80590  875 9750 0 0

Anti-leukotoxin Antibody Response in Mice

The mean absorbances per ml of serum, determined by ELISA, from micevaccinated with various leukotoxin polypeptides are shown in Table 5.

TABLE 5 Anti-leukotoxin antibody response in mice injected with variousleukotoxin preparations. D 46 Vaccine Preparations D 0 D 21 D 42(post-mortem) PBS 63.6^(a)  65.3^(a)  66.9^(a) 126.3^(d) BSBSE 52.9^(a) 90.2^(b) 179.4^(c)* 129.1^(d) SX 54.1^(a)  77.6^(ab) 186.4^(c)*144.5^(d) GAS 61.0^(a)  77.6^(ab)  97.1^(bc)* 109.6^(cd) SH 60.95^(a)101^(b)* 163.8^(c)* 130.0^(d) FINAL 63.9^(a)  66.2^(ab)  95.7^(bc)*121.7^(cd) BSBSE + GAS 79.7^(a)  82.5^(a) 161.1^(c)* 172.7^(cd)* ALLFIVE 66.1^(a)  98.9^(b)* 189^(c)* 219^(d)* Native Leukotoxin 59.6^(a)101.3^(b)* 235.5^(c)* 205.2^(d)* Culture Supernatant 76.4^(a) 105.7^(b)*205.4^(c)* 230.1^(cd)* Numbers with same superscripts were notsignificantly different from the ELISA values from mice belonging tosame group at a different sampling period. *Significantly different fromnegative control (PBS).

On day 21, mice vaccinated with affinity purified native leukotoxin,truncations BSBSE or SH, mixture of all five, or culture supernatant hadhigher antileukotoxin antibody levels compared to day 0. Serum collectedon day 21 from groups vaccinated with truncated polypeptide SH, mixtureof five truncations, native affinity purified leukotoxin or culturesupernatant, had significantly higher anti-leukotoxin antibody levelscompared to the control (PBS) group (p<0.01). There was no significantrise in the antibody levels on day 21 among mice vaccinated withtruncated polypeptides SX, GAS, FINAL, a combination of BSBSE and GAS orPBS. Mice belonging to group that was vaccinated with culturesupernatant, had significantly higher (P<0.01) antibody titers toleukotoxin than mice in other groups.

On day 42, there was a significant increase in antibody responsecompared to day 21 among mice vaccinated with all leukotoxinpreparations except GAS (P<0.01). Anti-leukotoxin antibody levels inserum from mice vaccinated with different leukotoxin polypeptides(including GAS) were significantly higher compared to the control. Theantibody response to a mixture of BSBSE+GAS was similar to BSBSE alonebut higher than GAS polypeptide. The antibody response to mixture of allfive was similar to BSBSE, SX, SH but higher than GAS or FINALpolypeptides. Mice vaccinated with affinity purified native leukotoxinhad the highest anti-leukotoxin antibody levels on day 42, followed bythose vaccinated with the culture supernatant and a mixture of all fiveoverlapping truncations. The truncated polypeptide GAS failed to raiseanti-leukotoxin antibody levels significantly after the secondvaccination compared to the day 21.

On day 46, 4 days after challenge with F. necrophorum (post-mortem),serum samples from mice vaccinated with leukotoxin polypeptides, BSBSE,SX, and SH, and affinity purified native leukotoxin had loweranti-leukotoxin antibody titers compared to day 42. Anti-leukotoxinantibody levels in mice vaccinated with GAS, FINAL, mixture of truncatedpolypeptides or culture supernatant had higher antibody levels comparedto day 42. Also, anti-leukotoxin antibody levels in mice in the controlgroup (vaccinated with PBS) on day 46 showed a significant increase thanserum collected before challenge (day 42). However, antibody levels inmice injected with BSBSE+GAS, mixture of all five, native leukotoxin andculture supernatant were higher than the control group.

Experimental Infection

Following the challenge with F. necrophorum, mice in all groupsexhibited acute shock within 24 hours perhaps induced by LPS. Mice inthe control or in the group vaccinated with inactivated culturesupernatant seemed to be affected most. The mice were listless,recumbent and did not seem to consume food or water. Mice vaccinatedwith various leukotoxin preparations recovered after 2 dayspost-challenge. Mice in the control group did not recover completelyfrom the symptoms of shock even by day 4 after challenge. Two mice inthe control group and one mouse in the group vaccinated with GASpolypeptide died about 36 hours after challenge. Pure cultures of F.necrophorum subsp. necrophorum were isolated from the heart blood of allthree mice.

Hepatic Pathology

Mice were euthanized 4 days after challenge and the internal organs wereexamined for abscesses. None of the mice vaccinated with leukotoxintruncation SH had any liver abscesses (Table 6).

TABLE 6 Mortality, liver abscess formation, weight of liver andbacterial load in liver in mice vaccinated with leukotoxin preparationsafter experimental challenge with Fusobacterium necrophorum. No. of miceLeukotoxin Number of with liver Average weight MPN counts preparationsdead mice abscess (%) of liver (g) in the liver Control 2/10 0/8(0)^(a)  1.86 5.3 × 10⁶  (PBS) BSBSE 0/10 1/10 (10) 1.29* 1.2 × 10³* SX0/10 5/10 (50) 1.39* 8.2 × 10⁵* GAS 1/10 3/9 (33)  1.32* 1.5 × 10⁶  SH0/10 0/10 (0)  1.20* 5.3 × 10²* FINAL 0/10 3/10 (30) 1.44* 6.8 × 10⁵*BSBSE + GAS 0/10 3/10 (30) 1.27* 1.4 × 10⁵* ALL FIVE 0/10 3/10 (30)1.33* 5.5 × 10⁵* Native 0/10 3/10 (30) 1.31* 5.9 × 10⁴* leukotoxinCulture 0/10 1/10 (10) 1.51* 1.6 × 10⁴* supernatant *Differs from thecontrol group (P < 0.01) ^(a)Livers lacked abscesses, but were highlycongested and icteric.

The eight mice that survived in the control group had highly congestedand icteric livers, but had no abscesses. Thirty percent of micevaccinated with affinity purified native leukotoxin, truncations GAS orFINAL, or mixtures (BSBSE and GAS, or all five truncations) had liverabscesses. Five out often mice vaccinated with leukotoxin truncatedpolypeptide SX developed liver abscesses. However, in the groupsvaccinated with the truncated leukotoxin polypeptide BSBSE orinactivated culture supernatant, only one out of 10 had liver abscesses.

The mean weight of livers from the control group was significantlyhigher than mean weights of livers from other groups. Livers from thegroup that received inactivated culture supernatant had the next biggestliver size. This correlated with the clinical signs of acute shockdisplayed by these two groups.

Enumeration of F. necrophorum in Liver Tissue

Fusobacterium necrophorum subsp. necrophorum was isolated fromhomogenized liver tissue and abscesses from all mice. The counts of F.necrophorum from livers of mice injected with any leukotoxin preparationwere lower (p<0.01) than the control (Table 6). Livers from micevaccinated with leukotoxin truncations BSBSE or SH showed significantlylower bacterial counts (p<0.01) than mice vaccinated with otherpreparations. Among leukotoxin truncations, SX showed least protectionfollowed by FINAL and GAS polypeptides as evidenced by the bacterialcounts in the livers of mice vaccinated with these polypeptides.Bacterial counts were considerably lower among groups vaccinated withmixtures of leukotoxin truncations (BSBSE and GAS or all fivetruncations), or affinity purified native leukotoxin as compared to thecontrol group but higher than SH, BSBSE or inactivated culturesupernatant (Table 6).

The five overlapping truncated leukotoxin polypeptides created allowedexpression of the entire leukotoxin gene without toxicity to the E. colihost cells. Primers for the amplification of various truncatedleukotoxin gene products were designed in such away that the expressedpolypeptides were not toxic to E. coil host cells, but were big enough(at least 30 kDa) to be a good immunogen. The nickel affinity columnpurified polypeptides were tested for purity in terms of contaminatingproteins or lipopolysaccharides by silver-staining the SDS-PAGEseparated proteins. Because all truncated polypeptides were purifiedunder denaturing conditions, they were not toxic as determined by theMTT assays. Fusobacterium necrophorum culture supernatant and affinitypurified native leukotoxin were inactivated with 0.3% formalin beforeinjection, thus were nontoxic.

Neutralization of toxicity of F. necrophorum leukotoxin against bovineperipheral PMNs by antiserum raised against BSBSE and GAS polypeptidessuggested that biologically important domains, such as those responsiblefor toxicity or host cell receptor binding was located in these regions.Therefore, a mixture of these two polypeptides (BSBSE+GAS) was also usedin a vaccine preparation in our challenge experiments with mice.

The significantly higher antibody levels noticed among groups vaccinatedwith preparations containing full-length leukotoxin proteins (nativeaffinity purified leukotoxin, culture supernatant, or a mixture ofrecombinant leukotoxin polypeptides containing all five truncations)maybe due to determinant spreading, or due to augmentation ofanti-leukotoxin antibody response by the presence of multipleimmunodominant epitopes on the leukotoxin protein. Truncated leukotoxinGAS produced a low antibody response. The high hydrophobicity of thispolypeptide maybe the reason for its reduced immunogenicity. Also, thewells in the ELISA plates were coated with native immunoaffinitypurified leukotoxin, and the domains represented by the GAS polypeptidecould possibly be hidden and not exposed for the antibodies against GASpolypeptide to bind.

Decrease in anti-leukotoxin antibody levels among various groups of miceon day 46 (4 days after experimental challenge with F. necrophorum)suggested neutralizing effect and clearance of toxin secreted by F.necrophorum used for experimental challenge by these antibodies. Purecultures of F. necrophorum subsp. necrophorum were isolated from theheart blood of the three mice (two from negative control group and onefrom group injected with GAS polypeptide) that died on day 2 afterchallenge, suggesting that death was due to septicemia induced by F.necrophorum. The hepatic tissue from the negative control group showedinflammation, congestion and icterus characteristic of an acute phaseresponse, but showed no abscesses.

Multiple responses including mortality, clinical signs, weights ofliver, presence of abscesses, and the bacterial load in liver wereconsidered to evaluate the effectiveness of various vaccine preparationsin providing immunity and protection against experimental challenge withF. necrophorum. Leukotoxin truncation SH was a very effective immunogenas evidenced by a rise in anti-leukotoxin antibody levels in serumsamples on day 21 or 42. Also, there were no mortality, hepaticinflammation or abscesses in mice vaccinated with this polypeptide afterexperimental challenge. The mean bacterial load in the livers of micefrom this group was the lowest (5.3×10²). Interestingly, leukotoxintruncated polypeptide SH did not induce neutralizing antibodies inrabbits. Production of high-affinity antibodies against certainimmunodominant domains that brings about effective opsonization andclearance of leukotoxin in an experimental challenge model may renderthis truncated polypeptide (SH) a protective antigen.

Vaccination with N-terminal truncation BSBSE or culture supernatantfollowed by experimental challenge with F. necrophorum caused nomortality, but livers were abscessed in 10% of the mice. Mice vaccinatedwith BSBSE, however, had less clinical signs of LPS induced shock aftervaccinations or challenge, lower liver weights and lowerhepatic-bacterial counts compared to mice vaccinated with inactivatedculture supernatant.

Native leukotoxin purified by immunoaffinity columns from F. necrophorumculture supernatant was the fourth best vaccine preparation (behind SH,BSBSE, and culture supernatant) in terms of serum antibody levels,protection against formation of liver abscess (30%), and number ofbacteria in the liver tissue. The vaccine consisting of a mixture of allfive recombinant truncated leukotoxin polypeptides also protected 70% ofmice from abscess formation and the bacterial counts in their hepatictissue were not significantly different from mice that were vaccinatedwith native leukotoxin.

Truncated polypeptide GAS, although it invoked neutralizing antibodiesin rabbits, was a poorer immunogen and protected 67% of the mice in itsgroup from formation of liver abscesses but one of the ten mice in thisgroup died after challenge. As mentioned above, this region couldcontain domain(s) of toxicological importance such as, target cellbinding, biological activities. However, multiple host-factors such as,availability of specific lymphocyte sub-population for clonal selection,type of helper T-cells stimulated, ability to invoke antibodies capableof opsonization, decide if an antibody response to a particular proteinis protective in the species of animal tested.

The truncated leukotoxin polypeptide SX provided least protection fromliver abscess formation. The number of bacteria in the hepatic tissue ofmice vaccinated with GAS or SX were significantly higher (P<0.01) thanin livers of mice vaccinated with SH, BSBSE, culture supernatant orfull-length native or recombinant leukotoxin (mixture of fivetruncations), but was lower than the mice in the negative control group.A mixture of BSBSE and GAS or the FINAL polypeptides provided only amediocre protection against experimental challenge. Polyclonal antiseraraised in rabbits against BSBSE or GAS neutralized the activity ofnative leukotoxin against PMNs used as target cells and were thus chosento be used in combination.

Recombinant truncated leukotoxin polypeptides SH and BSBSE providedsignificant protection in mice when used as a vaccine individually.Dilution of immunodominant and protective epitopes present within theseregions by including other truncated polypeptides as seen in vaccinepreparations containing affinity purified leukotoxin or combinations oftruncated leukotoxin polypeptides possibly caused a decrease in overallprotection. Further studies to test the effectiveness of leukotoxintruncations BSBSE and SH individually or in combination providingprotection against natural or experimental infections with F.necrophorum infections need to be carried out. This study providedfurther credence to the importance of leukotoxin as the major virulencefactor of F. necrophorum and the protein carries a domain(s) orepitope(s) that induces protective immunity against experimentalinfection. The vaccine that produced best antileukotoxin titer did notalways afford good protection against experimental infection. Therefore,certain epitopes maybe more important in conferring protective immunityto infection. The results of this study suggest that some of theseimportant epitopes reside on the BSBSE and SH polypeptides.

Discussion

Fusobacterium necrophorum subsp. necrophorum is isolated more often thansubsp. funduliforme from necrotic abscesses. The strains of subsp.necrophorum produces the high molecular weight leukotoxin in greaterquantities than strains of subsp. funduliforme. In this study, we havecloned the leukotoxin gene from the highly virulent F. necrophorumsubsp. necrophorum strain A25. The evidence that the lktA determinantencodes the leukotoxin is as follows: (1) the ORF encodes a 336 kDaprotein, a size consistent with previous studies of the toxin; (2) theprotein encoded by the recombinant lktA determinant is recognized byboth polyclonal and monoclonal antibodies raised against purifiedleukotoxin from F. necrophorum; (3) antisera raised against polypeptidesfrom the cloned lktA determinant recognized the native toxin in westernblots; (4) antisera raised against two of the truncated polypeptidesneutralized the toxic activity of the leukotoxin; and (5) therecombinant protein expressed in E. coli is relatively more toxic tobovine neutrophils as compared to bovine lymphocytes. These differingdegrees of toxicity toward neutrophils relative to lymphocytes is alsoobserved with leukotoxin that was affinity-purified from F. necrophorumculture supernatants.

The leukotoxin ORF is 9,726 base pairs long encoding a 3,241 amino acidprotein with an overall molecular mass of 335,956 daltons. The DNA anddeduced amino acid sequences were compared with sequences in Genbank butno significant (greater than 25% identity) similarities were found withother bacterial toxins. For example, the closest identity was found withHmwA from Haemophilus influenzae (22% or 356 out of 1,625 residues).Other similar homologies were found in SrpA from Streptococcus cristatus(17% or 388 out of 2,239 residues), OmpA from Ricketsia australis (21%or 321 out of 1,489 residues) and the 190 kDa surface antigen ofRickettsia ricketsii (21% or 379 out of 1,770 residues). Other Thus, theF. necrophorum leukotoxin appears to be distinct from all knownleukotoxins and RTX-type toxins. When the deduced amino acid sequence ofthe lktA region was subjected to the Kyte-Doolittle hydropathy analysis(FIG. 3), 14 sites of sufficient length and hydrophobic character to bepotential membrane spanning regions, were found. Upstream to theleukotoxin ORF is an open reading frame of at least 1.4 kb in length,which is in the same orientation. It encodes a protein that has somesequence identity to the heme-hemopexin utilization protein (UxuB) ofHaemophilus infuenzae.

Additionally, the protein is larger than any bacterial exotoxinsidentified to date and shows no sequence similarity to other knownleukotoxins. Thus, this protein may represent a new class of bacterialleukotoxins. The protein is unusual in that it is devoid of cysteine.This is not a characteristic of proteins from anaerobes, as evidenced bythe normal content of cysteine residues in the clostridial toxinsincluding Clostridium botulinum neurotoxin, Cl. difficile cytotoxin B,Cl. septicum alpha-toxin, and Cl. tetani tetanus toxin (Genbankaccession numbers AB037166, AB217292, D17668, and X06214, respectively).The leukotoxin protein has a sequence at its N-terminus that has theproperties of a signal sequence. This may indicate that the protein isexported across the cytoplasmic membrane in F. necrophorum in a Secpathway-dependent manner.

The DNA sequences flanking lktA suggests that this toxin gene maybe partof a multigene operon with at least one ORF upstream and anotherdownstream of this gene. The activity of the LktA protein expressed inE. coli indicates that the other proteins encoded in the putativeleukotoxin operon are not required to produce a biologically activetoxin. Their role may be in secretion of the toxin across thecytoplasmic and outer membranes of F. necrophorum into the culturefluid.

If the lktA determinant is part of an operon, it would be greater than12 kb in length. A dilemma with such a large operon might be toefficiently translate the messenger RNA species without prematuredissociation of ribosome from the message. A peculiarity in the clonedregion is an abundance of potential ribosome binding site sequences.Within the cloned region, there are 26 occurrences of GGAGG, which is aperfect match to the sequence at the 3′ end of the 16S rRNA. Thecomplementary sequence, CCTCC, which has the same G+C content but doesnot act as a ribosome binding site, is present only two times in thesequence. The abundance of the GGAGG sequence could provide translationreinforcement sequences to help ensure that a ribosome remainsassociated with the message and completes the translation of the ORFs.The abundance of the putative RBS sequence (GGAGG) is due to thepresence of di-glycine repeats in the amino acid sequence. The GGAglycine codon occurs 263 times in the leukotoxin ORF and 24 of the 26occurrences of GGAGG in the 11,130 bp sequenced to date correspond totandem repeats of this codon. This feature of the amino acid sequence inthe protein may provide the additional benefit of enabling moreefficient translation of the message.

Expressing the 3.5 kb sequence from the 5′ end of lktA caused immediatecessation of growth and lysis of E. coil carrying this recombinantexpression vector. Creation of overlapping truncations allowed theexpression of the entire leukotoxin gene without significant toxicity tothe E. coli host cells. Polyclonal antileukotoxin antiserum reactedstrongly to three truncated polypeptides (BSBSE, SX and FINAL) and moreweakly to the other two truncated polypeptides (GAS and SH) in westernblot analysis. This low reactivity was not due to poor immunogenicity ofthese relatively hydrophobic polypeptides, because both polypeptides(GAS and SH), produced high antibody titers in rabbits. Thus, it maybeen due to the tertiary folding pattern of leukotoxin under nativeconditions. The toxin being a secreted protein, would have itshydrophobic domains internalized when the protein was properly folded.The epitopes corresponding to these domains may not be as accessible tothe immune system. Antibodies against these epitopes would thus be underrepresented when the whole un-denatured toxin is used as the immunogen.Interestingly, antibodies to one of these polypeptides, GAS, wasneutralizing. Thus at least some of the critical epitopes are availablein the active toxin.

The intact leukotoxin gene was introduced into E. coli under the controlof the lac promoter. Inducible expression of full-length leukotoxinprotein was achieved without any recognizable toxicity to E. coli hostcells. Expression of the full-length leukotoxin instead of truncatedpolypeptides may allow correct folding of the toxin. This would resultin internalization of the hydrophobic domains with a correspondingreduction of toxicity in E. coli host cells. Both polyclonal andmonoclonal antibodies against native leukotoxin recognized a proteinspecies with a size consistent with that of the intact leukotoxin inwestern blot analysis of cell lysates of E. coli harboring pSN2000.Antibodies raised against all five truncated leukotoxin polypeptides,but not the upstream polypeptide, recognized full-length recombinantleukotoxin as well.

In order to determine the prevalence and heterogeneity of leukotoxingene in this species, 15 F. necrophorum strains belonging to subsp.necrophorum and subsp. funduliforme isolated from liver abscesses(opportunistic pathogen) or rumen contents (normal inhabitant) werescreened for lktA by Southern blotting. Strains belonging to F.necrophorum subsp. necrophorum, irrespective of its location ofisolation (liver abscess or ruminal contents) had similar hybridizingpatterns. Similarly, all strains of F. necrophorum subsp. funduliforme,irrespective of the site from which it was isolated had identicalhybridization patterns, but which differed from the subspeciesnecrophorum pattern. The difference in Southern blot hybridizationpatterns suggest that the disparity in levels of leukotoxin producedbetween the two subspecies may be due to differences in geneticorganization of the leukotoxin locus.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 15 <210> SEQ ID NO 1 <211> LENGTH: 3241<212> TYPE: PRT <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 1 Met Ser Gly Ile Lys Asn Asn Val Gln Arg Th#r Arg Lys Arg Ile Ser   1               5  #                 10 #                 15 Asp Ser Lys Lys Val Leu Met Ile Leu Gly Le#u Leu Ile Asn Thr Met              20      #             25     #             30 Thr Val Arg Ala Asn Asp Thr Ile Thr Ala Th#r Glu Asn Phe Gly Thr          35          #         40         #         45 Lys Ile Glu Lys Lys Asp Asn Val Tyr Asp Il#e Thr Thr Asn Lys Ile      50              #     55             #     60 Gln Gly Glu Asn Ala Phe Asn Ser Phe Asn Ar#g Phe Ala Leu Thr Glu  65                  # 70                 # 75                  # 80 Asn Asn Ile Ala Asn Leu Tyr Phe Gly Glu Ly#s Asn Ser Thr Gly Val                  85  #                 90 #                 95 Asn Asn Leu Phe Asn Phe Val Asn Gly Lys Il#e Glu Val Asp Gly Ile             100       #           105      #           110 Ile Asn Gly Ile Arg Glu Asn Lys Ile Gly Gl#y Asn Leu Tyr Phe Leu         115           #       120          #       125 Ser Ser Glu Gly Met Ala Val Gly Lys Asn Gl#y Val Ile Asn Ala Gly     130               #   135              #   140 Ser Phe His Ser Ile Ile Pro Lys Gln Asp As#p Phe Lys Lys Ala Leu 145                 1 #50                 1#55                 1 #60 Glu Glu Ala Lys His Gly Lys Val Phe Asn Gl#y Ile Ile Pro Val Asp                 165   #               170  #               175 Gly Lys Val Lys Ile Pro Leu Asn Pro Asn Gl#y Ser Ile Thr Val Glu             180       #           185      #           190 Gly Lys Ile Asn Ala Val Glu Gly Ile Gly Le#u Tyr Ala Ala Asp Ile         195           #       200          #       205 Arg Leu Lys Asp Thr Ala Ile Leu Lys Thr Gl#y Ile Thr Asp Phe Lys     210               #   215              #   220 Asn Leu Val Asn Ile Ser Asp Arg Ile Asn Se#r Gly Leu Thr Gly Asp 225                 2 #30                 2#35                 2 #40 Leu Lys Ala Thr Lys Thr Lys Ser Gly Asp Il#e Ile Leu Ser Ala His                 245   #               250  #               255 Ile Asp Ser Pro Gln Lys Ala Met Gly Lys As#n Ser Thr Val Gly Lys             260       #           265      #           270 Arg Ile Glu Glu Tyr Val Lys Gly Asn Thr Ly#s Ala Asn Ile Glu Ser         275           #       280          #       285 Asp Ala Val Leu Glu Ala Asp Gly Asn Ile Ly#s Ile Ser Ala Lys Ala     290               #   295              #   300 Thr Asn Gly Arg Phe Ile Lys Lys Glu Gly Gl#u Lys Glu Thr Tyr Asn 305                 3 #10                 3#15                 3 #20 Thr Pro Leu Ser Leu Ser Asp Val Glu Ala Se#r Val Arg Val Asn Lys                 325   #               330  #               335 Gly Lys Val Ile Gly Lys Asn Val Asp Ile Th#r Ala Glu Ala Lys Asn             340       #           345      #           350 Phe Tyr Asp Ala Thr Leu Val Thr Lys Leu Al#a Lys His Ser Phe Ser         355           #       360          #       365 Phe Val Thr Gly Ser Ile Ser Pro Ile Asn Le#u Asn Gly Phe Leu Gly     370               #   375              #   380 Leu Leu Thr Ser Lys Ser Ser Val Val Ile Gl#y Lys Asp Ala Lys Val 385                 3 #90                 3#95                 4 #00 Glu Ala Thr Glu Gly Lys Ala Asn Ile His Se#r Tyr Ser Gly Val Arg                 405   #               410  #               415 Ala Thr Met Gly Ala Ala Thr Ser Pro Leu Ly#s Ile Thr Asn Leu Tyr             420       #           425      #           430 Leu Glu Lys Ala Asn Gly Lys Leu Leu Ser Il#e Gly Ala Gly Tyr Ile         435           #       440          #       445 Ser Ala Lys Ser Asn Ser Asn Val Thr Ile Gl#u Gly Glu Val Lys Ser     450               #   455              #   460 Lys Gly Arg Ala Asp Ile Thr Ser Lys Ser Gl#u Asn Thr Ile Asp Ala 465                 4 #70                 4#75                 4 #80 Ser Val Ser Val Gly Thr Met Arg Asp Ser As#n Lys Val Ala Leu Ser                 485   #               490  #               495 Val Leu Val Thr Glu Gly Glu Asn Lys Ser Se#r Val Lys Ile Ala Lys             500       #           505      #           510 Gly Ala Lys Val Glu Ser Glu Thr Asp Asp Va#l Asn Val Arg Ser Glu         515           #       520          #       525 Ala Ile Asn Ser Ile Arg Ala Ala Val Lys Gl#y Gly Leu Gly Asp Ser     530               #   535              #   540 Gly Asn Gly Val Val Ala Ala Asn Ile Ser As#n Tyr Asn Ala Ser Ser 545                 5 #50                 5#55                 5 #60 Arg Ile Asp Val Asp Gly Tyr Leu His Ala Ly#s Lys Arg Leu Asn Val                 565   #               570  #               575 Glu Ala His Asn Ile Thr Lys Asn Ser Val Le#u Gln Thr Gly Ser Asp             580       #           585      #           590 Leu Gly Thr Ser Lys Phe Met Asn Asp His Va#l Tyr Glu Ser Gly His         595           #       600          #       605 Leu Lys Ser Ile Leu Asp Ala Ile Lys Gln Ar#g Phe Gly Gly Asp Ser     610               #   615              #   620 Val Asn Glu Glu Ile Lys Asn Lys Leu Thr As#n Leu Phe Ser Val Gly 625                 6 #30                 6#35                 6 #40 Val Ser Ala Thr Ile Ala Asn His Asn Asn Se#r Ala Ser Val Ala Ile                 645   #               650  #               655 Gly Glu Ser Gly Arg Leu Ser Ser Gly Val Gl#u Gly Ser Asn Val Arg             660       #           665      #           670 Ala Leu Asn Glu Ala Gln Asn Leu Arg Ala Th#r Thr Ser Ser Gly Ser         675           #       680          #       685 Val Ala Val Arg Lys Glu Glu Lys Lys Lys Le#u Ile Gly Asn Ala Ala     690               #   695              #   700 Val Phe Tyr Gly Asn Tyr Lys Asn Asn Ala Se#r Val Thr Ile Ala Asp 705                 7 #10                 7#15                 7 #20 His Ala Glu Leu Val Ser Glu Gly Lys Ile As#p Ile Asn Ser Glu Asn                 725   #               730  #               735 Lys Ile Glu Tyr Lys Asn Pro Ser Lys Met Al#a Lys Ser Val Ile Asp             740       #           745      #           750 Lys Leu Glu Leu Leu Lys Arg Ala Phe Gly Ly#s Glu Thr Lys Thr Pro         755           #       760          #       765 Glu Tyr Asp Pro Lys Asp Ile Glu Ser Ile Gl#u Lys Leu Leu Asn Ala     770               #   775              #   780 Phe Ser Glu Lys Leu Asp Gly Lys Pro Glu Le#u Leu Leu Asn Gly Glu 785                 7 #90                 7#95                 8 #00 Arg Met Thr Ile Ile Leu Pro Asp Gly Thr Se#r Lys Thr Gly Thr Ala                 805   #               810  #               815 Ile Glu Ile Ala Asn Tyr Val Gln Gly Glu Me#t Lys Lys Leu Glu Glu             820       #           825      #           830 Lys Leu Pro Lys Gly Phe Lys Ala Phe Ser Gl#u Gly Leu Ser Gly Leu         835           #       840          #       845 Ile Lys Glu Thr Leu Asn Phe Thr Gly Val Gl#y Asn Tyr Ala Asn Phe     850               #   855              #   860 His Thr Phe Thr Ser Ser Gly Ala Asn Gly Gl#u Arg Asp Val Ser Ser 865                 8 #70                 8#75                 8 #80 Val Gly Gly Ala Val Ser Trp Val Glu Gln Gl#u Asn Tyr Ser Lys Val                 885   #               890  #               895 Ser Val Gly Lys Gly Ala Lys Leu Ala Ala Ly#s Lys Asp Leu Asn Ile             900       #           905      #           910 Lys Ala Ile Asn Lys Ala Glu Thr Val Asn Le#u Val Gly Asn Ile Gly         915           #       920          #       925 Leu Ala Arg Ser Ser Thr Ser Gly Ser Ala Va#l Gly Gly Arg Leu Asn     930               #   935              #   940 Val Gln Arg Ser Lys Asn Ser Ala Ile Val Gl#u Ala Lys Glu Lys Ala 945                 9 #50                 9#55                 9 #60 Glu Leu Ser Gly Glu Asn Ile Asn Ala Asp Al#a Leu Asn Arg Leu Phe                 965   #               970  #               975 His Val Ala Gly Ser Phe Asn Gly Gly Ser Gl#y Gly Asn Ala Ile Asn             980       #           985      #           990 Gly Met Gly Ser Tyr Ser Gly Gly Ile Ser Ly#s Ala Arg Val Ser Ile         995           #      1000           #     1005 Asp Asp Glu Ala Tyr Leu Lys Ala Asn Lys Ly#s Ile Ala Leu Asn Ser    1010               #  1015               # 1020 Lys Asn Asp Thr Ser Val Trp Asn Ala Ala Gl #y Ser Ala Gly Ile Gly1025               1030  #               1035   #              1040Thr Lys Asn Ala Ala Val Gly Val Ala Val Al #a Val Asn Asp Tyr Asp               1045   #              1050    #             1055Ile Ser Asn Lys Ala Ser Ile Glu Asp Asn As #p Glu Gly Gln Ser Lys           1060       #          1065        #         1070Tyr Asp Lys Asn Lys Asp Asp Glu Val Thr Va #l Thr Ala Glu Ser Leu       1075           #      1080            #     1085Glu Val Asp Ala Lys Thr Thr Gly Thr Ile As #n Ser Ile Ser Val Ala   1090               #  1095                # 1100Gly Gly Ile Asn Lys Val Gly Ser Lys Pro Se #r Glu Glu Lys Pro Lys1105               1110  #               1115   #              1120Ser Glu Glu Arg Pro Glu Gly Phe Phe Gly Ly #s Ile Gly Asn Lys Val               1125   #              1130    #             1135Asp Ser Val Lys Asn Lys Ile Thr Asp Ser Me #t Asp Ser Leu Thr Glu           1140       #          1145        #         1150Lys Ile Thr Asn Tyr Ile Ser Glu Gly Val Ly #s Lys Ala Gly Asn Leu       1155           #      1160            #     1165Pro Ser Asn Val Ser His Thr Pro Asp Lys Gl #y Pro Ser Phe Ser Leu   1170               #  1175                # 1180Gly Ala Ser Gly Ser Val Ser Phe Asn Asn Il #e Lys Lys Glu Thr Ser1185               1190  #               1195   #              1200Ala Val Val Asp Gly Val Lys Ile Asn Leu Ly #s Gly Ala Asn Lys Lys               1205   #              1210    #             1215Val Glu Val Thr Ser Ser Asp Ser Thr Phe Va #l Gly Ala Trp Gly Gly           1220       #          1225        #         1230Ser Ala Ala Leu Gln Trp Asn His Ile Gly Se #r Gly Asn Ser Asn Ile       1235           #      1240            #     1245Ser Ala Gly Leu Ala Gly Ala Ala Ala Val As #n Asn Ile Gln Ser Lys   1250               #  1255                # 1260Thr Ser Ala Leu Val Lys Asn Ser Asp Ile Ar #g Asn Ala Asn Lys Phe1265               1270  #               1275   #              1280Lys Val Asn Ala Leu Ser Gly Gly Thr Gln Va #l Ala Ala Gly Ala Gly               1285   #              1290    #             1295Leu Glu Ala Val Lys Glu Ser Gly Gly Gln Gl #y Lys Ser Tyr Leu Leu           1300       #          1305        #         1310Gly Thr Ser Ala Ser Ile Asn Leu Val Asn As #n Glu Val Ser Ala Lys       1315           #      1320            #     1325Ser Glu Asn Asn Thr Val Ala Gly Glu Ser Gl #u Ser Gln Lys Met Asp   1330               #  1335                # 1340Val Asp Val Thr Ala Tyr Gln Ala Asp Thr Gl #n Val Thr Gly Ala Leu1345               1350  #               1355   #              1360Asn Leu Gln Ala Gly Lys Ser Asn Gly Thr Va #l Gly Ala Thr Val Thr               1365   #              1370    #             1375Val Ala Lys Leu Asn Asn Lys Val Asn Ala Se #r Ile Ser Gly Gly Arg           1380       #          1385        #         1390Tyr Thr Asn Val Asn Arg Ala Asp Ala Lys Al #a Leu Leu Ala Thr Thr       1395           #      1400            #     1405Gln Val Thr Ala Ala Val Thr Thr Gly Gly Th #r Ile Ser Ser Gly Ala   1410               #  1415                # 1420Gly Leu Gly Asn Tyr Gln Gly Ala Val Ser Va #l Asn Lys Ile Asp Asn1425               1430  #               1435   #              1440Asp Val Glu Ala Ser Val Asp Lys Ser Ser Il #e Glu Gly Ala Asn Glu               1445   #              1450    #             1455Ile Asn Val Ile Ala Lys Asp Val Lys Gly Se #r Ser Asp Leu Ala Lys           1460       #          1465        #         1470Glu Tyr Gln Ala Leu Leu Asn Gly Lys Asp Ly #s Lys Tyr Leu Glu Asp       1475           #      1480            #     1485Arg Gly Ile Asn Thr Thr Gly Asn Gly Tyr Ty #r Thr Lys Glu Gln Leu   1490               #  1495                # 1500Glu Lys Ala Lys Lys Lys Glu Gly Ala Val Il #e Val Asn Ala Ala Leu1505               1510  #               1515   #              1520Ser Val Ala Gly Thr Asp Lys Ser Ala Gly Gl #y Val Ala Ile Ala Val               1525   #              1530    #             1535Asn Thr Val Lys Asn Lys Phe Lys Ala Glu Le #u Ser Gly Ser Asn Lys           1540       #          1545        #         1550Glu Ala Gly Glu Asp Lys Ile His Ala Lys Hi #s Val Asn Val Glu Ala       1555           #      1560            #     1565Lys Ser Ser Thr Val Val Val Asn Ala Ala Se #r Gly Leu Ala Ile Ser   1570               #  1575                # 1580Lys Asp Ala Phe Ser Gly Met Gly Ser Gly Al #a Trp Gln Asp Leu Ser1585               1590  #               1595   #              1600Asn Asp Thr Ile Ala Lys Val Asp Lys Gly Ar #g Ile Ser Ala Asp Ser               1605   #              1610    #             1615Leu Asn Val Asn Ala Asn Asn Ser Ile Leu Gl #y Val Asn Val Ala Gly           1620       #          1625        #         1630Thr Ile Ala Gly Ser Leu Ser Thr Ala Val Gl #y Ala Ala Phe Ala Asn       1635           #      1640            #     1645Asn Thr Leu His Asn Lys Thr Ser Ala Leu Il #e Thr Gly Thr Lys Val   1650               #  1655                # 1660Asn Pro Phe Ser Gly Lys Asn Thr Lys Val As #n Val Gln Ala Leu Asn1665               1670  #               1675   #              1680Asp Ser His Ile Thr Asn Val Ser Ala Gly Gl #y Ala Ala Ser Ile Lys               1685   #              1690    #             1695Gln Ala Gly Ile Gly Gly Met Val Ser Val As #n Arg Gly Ser Asp Glu           1700       #          1705        #         1710Thr Glu Ala Leu Val Ser Asp Ser Glu Phe Gl #u Gly Val Ser Ser Phe       1715           #      1720            #     1725Asn Val Asp Ala Lys Asp Gln Lys Thr Ile As #n Thr Ile Ala Gly Asn   1730               #  1735                # 1740Ala Asn Gly Gly Lys Ala Ala Gly Val Gly Al #a Thr Val Ala His Thr1745               1750  #               1755   #              1760Asn Ile Gly Lys Gln Ser Val Ile Ala Ile Va #l Lys Asn Ser Lys Ile               1765   #              1770    #             1775Thr Thr Ala Asn Asp Gln Asp Arg Lys Asn Il #e Asn Val Thr Ala Lys           1780       #          1785        #         1790Asp Tyr Thr Met Thr Asn Thr Ile Ala Val Gl #y Val Gly Gly Ala Lys       1795           #      1800            #     1805Gly Ala Ser Val Gln Gly Ala Ser Ala Ser Th #r Thr Leu Asn Lys Thr   1810               #  1815                # 1820Val Ser Ser His Val Asp Gln Thr Asp Ile As #p Lys Asp Leu Glu Glu1825               1830  #               1835   #              1840Glu Asn Asn Gly Asn Lys Glu Lys Ala Asn Va #l Asn Val Leu Ala Glu               1845   #              1850    #             1855Asn Thr Ser Gln Val Val Thr Asn Ala Thr Va #l Leu Ser Gly Ala Ser           1860       #          1865        #         1870Gly Gln Ala Ala Val Gly Ala Gly Val Ala Va #l Asn Lys Ile Thr Gln       1875           #      1880            #     1885Asn Thr Ser Ala His Ile Lys Asn Ser Thr Gl #n Asn Val Arg Asn Ala   1890               #  1895                # 1900Leu Val Lys Ser Lys Ser His Ser Ser Ile Ly #s Thr Ile Gly Ile Gly1905               1910  #               1915   #              1920Ala Gly Val Gly Ala Gly Gly Ala Gly Val Th #r Gly Ser Val Ala Val               1925   #              1930    #             1935Asn Lys Ile Val Asn Asn Thr Ile Ala Glu Le #u Asn His Ala Lys Ile           1940       #          1945        #         1950Thr Ala Lys Gly Asn Val Gly Val Ile Thr Gl #u Ser Asp Ala Val Ile       1955           #      1960            #     1965Ala Asn Tyr Ala Gly Thr Val Ser Gly Val Al #a Arg Ala Ala Ile Gly   1970               #  1975                # 1980Ala Ser Thr Ser Val Asn Glu Ile Thr Gly Se #r Thr Lys Ala Tyr Val1985               1990  #               1995   #              2000Lys Asp Ser Thr Val Ile Ala Lys Glu Glu Th #r Asp Asp Tyr Ile Thr               2005   #              2010    #             2015Thr Gln Gly Gln Val Asp Lys Val Val Asp Ly #s Val Phe Lys Asn Leu           2020       #          2025        #         2030Asn Ile Asn Glu Asp Leu Ser Gln Lys Arg Ly #s Ile Ser Asn Lys Lys       2035           #      2040            #     2045Gly Phe Val Thr Asn Ser Ser Ala Thr His Th #r Leu Lys Ser Leu Leu   2050               #  2055                # 2060Ala Asn Ala Ala Gly Ser Gly Gln Ala Gly Va #l Ala Gly Thr Val Asn2065               2070  #               2075   #              2080Ile Asn Lys Val Tyr Gly Glu Thr Glu Ala Le #u Val Glu Asn Ser Ile               2085   #              2090    #             2095Leu Asn Ala Lys His Tyr Ser Val Lys Ser Gl #y Asp Tyr Thr Asn Ser           2100       #          2105        #         2110Ile Gly Val Val Gly Ser Val Gly Val Gly Gl #y Asn Val Gly Val Gly       2115           #      2120            #     2125Ala Ser Ser Asp Thr Asn Ile Ile Lys Arg As #n Thr Lys Thr Arg Val   2130               #  2135                # 2140Gly Lys Thr Thr Met Ser Asp Glu Gly Phe Gl #y Glu Glu Ala Glu Ile2145               2150  #               2155   #              2160Thr Ala Asp Ser Lys Gln Gly Ile Ser Ser Ph #e Gly Val Gly Val Ala               2165   #              2170    #             2175Ala Ala Gly Val Gly Ala Gly Val Ala Gly Th #r Val Ser Val Asn Gln           2180       #          2185        #         2190Phe Ala Gly Lys Thr Glu Val Asp Val Glu Gl #u Ala Lys Ile Leu Val       2195           #      2200            #     2205Lys Lys Ala Glu Ile Thr Ala Lys Arg Tyr Se #r Ser Val Ala Ile Gly   2210               #  2215                # 2220Asn Ala Ala Val Gly Val Ala Ala Lys Gly Al #a Gly Ile Gly Ala Ala2225               2230  #               2235   #              2240Val Ala Val Thr Lys Asp Glu Ser Asn Thr Ar #g Ala Arg Val Lys Asn               2245   #              2250    #             2255Ser Lys Ile Met Thr Arg Asn Lys Leu Asp Va #l Ile Ala Glu Asn Glu           2260       #          2265        #         2270Ile Lys Ser Gly Thr Gly Ile Gly Ser Ala Gl #y Ala Gly Ile Leu Ala       2275           #      2280            #     2285Ala Gly Val Ser Gly Val Val Ser Val Asn As #n Ile Ala Asn Lys Val   2290               #  2295                # 2300Glu Thr Asp Ile Asp His Ser Thr Leu His Se #r Ser Thr Asp Val Asn2305               2310  #               2315   #              2320Val Lys Ala Leu Asn Lys Ile Ser Asn Ser Le #u Thr Ala Gly Gly Gly               2325   #              2330    #             2335Ala Ala Gly Leu Ala Ala Val Thr Gly Val Va #l Ser Val Asn Thr Ile           2340       #          2345        #         2350Asn Ser Ser Val Ile Ala Arg Val His Asn As #n Ser Asp Leu Thr Ser       2355           #      2360            #     2365Val Arg Glu Lys Val Asn Val Thr Ala Lys Gl #u Glu Lys Asn Ile Lys   2370               #  2375                # 2380Gln Thr Ala Ala Asn Ala Gly Ile Gly Gly Al #a Ala Ile Gly Ala Asn2385               2390  #               2395   #              2400Val Leu Val Asn Asn Phe Gly Thr Ala Val Gl #u Asp Arg Lys Asn Ser               2405   #              2410    #             2415Glu Gly Lys Gly Thr Glu Val Leu Lys Thr Le #u Asp Glu Val Asn Lys           2420       #          2425        #         2430Glu Gln Asp Lys Lys Val Asn Asp Ala Thr Ly #s Lys Ile Leu Gln Ser       2435           #      2440            #     2445Ala Gly Ile Ser Thr Glu Asp Thr Ser Val Ly #s Ala Asp Arg Gly Asp   2450               #  2455                # 2460Thr Gln Gly Glu Gly Ile Lys Ala Ile Val Ly #s Thr Ser Asp Ile Ile2465               2470  #               2475   #              2480Gly Lys Asn Val Asp Ile Thr Thr Glu Asp Ly #s Asn Asn Ile Thr Ser               2485   #              2490    #             2495Thr Gly Gly Leu Gly Thr Ala Gly Leu Ala Se #r Ala Ser Gly Thr Val           2500       #          2505        #         2510Ala Val Thr Asn Ile Lys Arg Asn Ser Gly Va #l Thr Val Glu Asn Ser       2515           #      2520            #     2525Phe Val Lys Ala Ala Glu Lys Val Asn Val Ar #g Ser Asp Ile Thr Gly   2530               #  2535                # 2540Asn Val Ala Leu Thr Ala Tyr Gln Gly Pro Va #l Gly Ala Leu Gly Ile2545               2550  #               2555   #              2560Gly Ala Ala Tyr Ala Glu Leu Asn Ser Asn Gl #y Arg Ser Asn Ile Ser               2565   #              2570    #             2575Ile Lys Asn Ser Lys Leu Leu Gly Lys Asn Il #e Asp Val Ile Val Lys           2580       #          2585        #         2590Asp Lys Ser Glu Leu Arg Ala Glu Ala Lys Gl #y Leu Thr Val Gly Ala       2595           #      2600            #     2605Val Ala Ala Gly Ala Ile Ile Ser Lys Ala Ly #s Asn Glu Met Asn Ser   2610               #  2615                # 2620Glu Val Glu Ile Glu Lys Ser Ile Phe Asn Gl #u Glu Asn Arg Val Thr2625               2630  #               2635   #              2640Ser Pro Ser Lys Gly Ile Gly Arg Glu Ile As #n Val Lys Val Glu Lys               2645   #              2650    #             2655Glu Asn Arg Val Thr Ala Glu Ser Gln Gly Al #a Ser Val Gly Ala Val           2660       #          2665        #         2670Ala Gly Ala Gly Ile Ile Ser Glu Ala Lys As #p Ala Gly Ser Ser Tyr       2675           #      2680            #     2685Leu Lys Val Ser Thr Lys Ser Gly Arg Ser Il #e Phe His Ala Asp Asn   2690               #  2695                # 2700Val Asn Met Glu Ala Thr His Lys Met Lys Va #l Thr Ala Val Ser Lys2705               2710  #               2715   #              2720Ala Val Thr Gly Ser Val Leu Gly Gly Val Gl #y Val Thr Lys Ala Glu               2725   #              2730    #             2735Ala Thr Ala Ala Gly Lys Thr Met Val Glu Va #l Glu Glu Gly Asn Leu           2740       #          2745        #         2750Phe Arg Thr Asn Arg Leu Asn Ala Ile Ser Ly #s Val Glu Gly Leu Asp       2755           #      2760            #     2765Glu Asp Lys Val Thr Ala Lys Ser Ser Val Va #l Ser Gly Asn Gly Gly   2770               #  2775                # 2780Gly Ile Ala Gly Ala Gly Val Asn Thr Ser Th #r Ala Gln Ser Asn Thr2785               2790  #               2795   #              2800Glu Ser Val Val Arg Leu Arg Lys Gln Asp Ty #r Glu Asn Asn Asp Tyr               2805   #              2810    #             2815Thr Lys Lys Tyr Ile Ser Glu Val Asn Ala Le #u Ala Leu Asn Asp Thr           2820       #          2825        #         2830Lys Asn Glu Ala Asn Ile Glu Ser Leu Ala Va #l Ala Gly Val His Ala       2835           #      2840            #     2845Gln Gly Thr Asn Lys Ala Phe Thr Arg Ser As #n Lys Leu Thr Ser Thr   2850               #  2855                # 2860Thr Val Asn Gly Gly Asn Val Ser Gln Leu Ar #g Ala Lys Ala Leu Ala2865               2870  #               2875   #              2880Lys Asn Glu Asn Tyr Gly Asn Val Lys Gly Th #r Gly Gly Ala Leu Val               2885   #              2890    #             2895Gly Ala Glu Thr Ala Ala Val Glu Asn Tyr Th #r Lys Ser Thr Thr Gly           2900       #          2905        #         2910Ala Leu Val Ala Gly Asn Trp Glu Ile Gly As #p Lys Leu Glu Thr Ile       2915           #      2920            #     2925Ala Arg Asp Asn Thr Ile Val Arg Val Asn Gl #y Asp Gly Thr Lys Gly   2930               #  2935                # 2940Gly Leu Val Gly Lys Asn Gly Ile Ser Val Ly #s Asn Thr Ile Ser Gly2945               2950  #               2955   #              2960Glu Thr Lys Ser Ser Ile Glu Asp Lys Ala Ar #g Ile Val Gly Thr Gly               2965   #              2970    #             2975Ser Val Asn Val Asp Ala Leu Asn Glu Leu As #p Val Asp Leu Gln Gly           2980       #          2985        #         2990Lys Ser Gly Gly Tyr Gly Gly Ile Gly Ile Gl #y Asn Val Asp Val Asn       2995           #      3000            #     3005Asn Val Ile Lys Lys Asn Val Glu Ala Lys Il #e Gly Arg His Ala Ile   3010               #  3015                # 3020Val Glu Thr Thr Gly Lys Gln Glu Tyr Gln Al #a Phe Thr Arg Ala Lys3025               3030  #               3035   #              3040Val Asn Ile Leu Gly Lys Gly Asp Ala Ala Al #a Ala Ala Ala Ile Ser               3045   #              3050    #             3055Asn Val His Ile Ser Asn Glu Met Asp Ile Ly #s Asn Leu Ala Lys Gln           3060       #          3065        #         3070Tyr Ala Ser Ser Gln Leu Ile Thr Lys Asn Se #r Lys Asn Asn Ile Thr       3075           #      3080            #     3085Leu Ala Ser Ser Ser Glu Ser Asn Val Asn Va #l His Gly Val Ala Glu   3090               #  3095                # 3100Ala Arg Gly Ala Gly Ala Lys Ala Thr Val Se #r Val Lys Asn Gln Ile3105               3110  #               3115   #              3120Asn Arg Thr Asn Asn Val Asp Leu Ala Gly Ly #s Ile Lys Thr Glu Gly               3125   #              3130    #             3135Asn Ile Asn Val Tyr Ala Gly Tyr Asp Lys As #n Tyr Asn Ile Ser Lys           3140       #          3145        #         3150Thr Asn Ser Lys Ala Ile Ala Asp Ala Lys Se #r His Ala Ala Ala Ala       3155           #      3160            #     3165Ser Ala Thr Ala Thr Ile Glu Lys Asn Glu Va #l Lys Phe Asn Asn Ala   3170               #  3175                # 3180Ile Arg Glu Phe Lys Asn Asn Leu Ala Arg Le #u Glu Gly Lys Ala Asn3185               3190  #               3195   #              3200Lys Lys Thr Ser Val Gly Ser Asn Gln Val As #p Trp Tyr Thr Asp Lys               3205   #              3210    #             3215Tyr Thr Trp His Ser Ser Glu Lys Ala Tyr Ly #s Lys Leu Thr Tyr Gln           3220       #          3225        #         3230Ser Lys Arg Gly Glu Lys Gly Lys Lys        3235           #      3240<210> SEQ ID NO 2 <211> LENGTH: 369 <212> TYPE: PRT<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 2Met Ser Gly Ile Lys Asn Asn Val Gln Arg Th #r Arg Lys Arg Ile Ser  1               5  #                 10  #                 15Asp Ser Lys Lys Val Leu Met Ile Leu Gly Le #u Leu Ile Asn Thr Met             20      #             25      #             30Thr Val Arg Ala Asn Asp Thr Ile Thr Ala Th #r Glu Asn Phe Gly Thr         35          #         40          #         45Lys Ile Glu Lys Lys Asp Asn Val Tyr Asp Il #e Thr Thr Asn Lys Ile     50              #     55              #     60Gln Gly Glu Asn Ala Phe Asn Ser Phe Asn Ar #g Phe Ala Leu Thr Glu 65                  # 70                  # 75                  # 80Asn Asn Ile Ala Asn Leu Tyr Phe Gly Glu Ly #s Asn Ser Thr Gly Val                 85  #                 90  #                 95Asn Asn Leu Phe Asn Phe Val Asn Gly Lys Il #e Glu Val Asp Gly Ile            100       #           105       #           110Ile Asn Gly Ile Arg Glu Asn Lys Ile Gly Gl #y Asn Leu Tyr Phe Leu        115           #       120           #       125Ser Ser Glu Gly Met Ala Val Gly Lys Asn Gl #y Val Ile Asn Ala Gly    130               #   135               #   140Ser Phe His Ser Ile Ile Pro Lys Gln Asp As #p Phe Lys Lys Ala Leu145                 1 #50                 1 #55                 1 #60Glu Glu Ala Lys His Gly Lys Val Phe Asn Gl #y Ile Ile Pro Val Asp                165   #               170   #               175Gly Lys Val Lys Ile Pro Leu Asn Pro Asn Gl #y Ser Ile Thr Val Glu            180       #           185       #           190Gly Lys Ile Asn Ala Val Glu Gly Ile Gly Le #u Tyr Ala Ala Asp Ile        195           #       200           #       205Arg Leu Lys Asp Thr Ala Ile Leu Lys Thr Gl #y Ile Thr Asp Phe Lys    210               #   215               #   220Asn Leu Val Asn Ile Ser Asp Arg Ile Asn Se #r Gly Leu Thr Gly Asp225                 2 #30                 2 #35                 2 #40Leu Lys Ala Thr Lys Thr Lys Ser Gly Asp Il #e Ile Leu Ser Ala His                245   #               250   #               255Ile Asp Ser Pro Gln Lys Ala Met Gly Lys As #n Ser Thr Val Gly Lys            260       #           265       #           270Arg Ile Glu Glu Tyr Val Lys Gly Asn Thr Ly #s Ala Asn Ile Glu Ser        275           #       280           #       285Asp Ala Val Leu Glu Ala Asp Gly Asn Ile Ly #s Ile Ser Ala Lys Ala    290               #   295               #   300Thr Asn Gly Arg Phe Ile Lys Lys Glu Gly Gl #u Lys Glu Thr Tyr Asn305                 3 #10                 3 #15                 3 #20Thr Pro Leu Ser Leu Ser Asp Val Glu Ala Se #r Val Arg Val Asn Lys                325   #               330   #               335Gly Lys Val Ile Gly Lys Asn Val Asp Ile Th #r Ala Glu Ala Lys Asn            340       #           345       #           350Phe Tyr Asp Ala Thr Leu Val Thr Lys Leu Al #a Lys His Ser Phe Ser        355           #       360           #       365 Phe<210> SEQ ID NO 3 <211> LENGTH: 927 <212> TYPE: PRT<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 3Gly Arg Phe Ile Lys Lys Glu Gly Glu Lys Gl #u Thr Tyr Asn Thr Pro  1               5  #                 10  #                 15Leu Ser Leu Ser Asp Val Glu Ala Ser Val Ar #g Val Asn Lys Gly Lys             20      #             25      #             30Val Ile Gly Lys Asn Val Asp Ile Thr Ala Gl #u Ala Lys Asn Phe Tyr         35          #         40          #         45Asp Ala Thr Leu Val Thr Lys Leu Ala Lys Hi #s Ser Phe Ser Phe Val     50              #     55              #     60Thr Gly Ser Ile Ser Pro Ile Asn Leu Asn Gl #y Phe Leu Gly Leu Leu 65                  # 70                  # 75                  # 80Thr Ser Lys Ser Ser Val Val Ile Gly Lys As #p Ala Lys Val Glu Ala                 85  #                 90  #                 95Thr Glu Gly Lys Ala Asn Ile His Ser Tyr Se #r Gly Val Arg Ala Thr            100       #           105       #           110Met Gly Ala Ala Thr Ser Pro Leu Lys Ile Th #r Asn Leu Tyr Leu Glu        115           #       120           #       125Lys Ala Asn Gly Lys Leu Leu Ser Ile Gly Al #a Gly Tyr Ile Ser Ala    130               #   135               #   140Lys Ser Asn Ser Asn Val Thr Ile Glu Gly Gl #u Val Lys Ser Lys Gly145                 1 #50                 1 #55                 1 #60Arg Ala Asp Ile Thr Ser Lys Ser Glu Asn Th #r Ile Asp Ala Ser Val                165   #               170   #               175Ser Val Gly Thr Met Arg Asp Ser Asn Lys Va #l Ala Leu Ser Val Leu            180       #           185       #           190Val Thr Glu Gly Glu Asn Lys Ser Ser Val Ly #s Ile Ala Lys Gly Ala        195           #       200           #       205Lys Val Glu Ser Glu Thr Asp Asp Val Asn Va #l Arg Ser Glu Ala Ile    210               #   215               #   220Asn Ser Ile Arg Ala Ala Val Lys Gly Gly Le #u Gly Asp Ser Gly Asn225                 2 #30                 2 #35                 2 #40Gly Val Val Ala Ala Asn Ile Ser Asn Tyr As #n Ala Ser Ser Arg Ile                245   #               250   #               255Asp Val Asp Gly Tyr Leu His Ala Lys Lys Ar #g Leu Asn Val Glu Ala            260       #           265       #           270His Asn Ile Thr Lys Asn Ser Val Leu Gln Th #r Gly Ser Asp Leu Gly        275           #       280           #       285Thr Ser Lys Phe Met Asn Asp His Val Tyr Gl #u Ser Gly His Leu Lys    290               #   295               #   300Ser Ile Leu Asp Ala Ile Lys Gln Arg Phe Gl #y Gly Asp Ser Val Asn305                 3 #10                 3 #15                 3 #20Glu Glu Ile Lys Asn Lys Leu Thr Asn Leu Ph #e Ser Val Gly Val Ser                325   #               330   #               335Ala Thr Ile Ala Asn His Asn Asn Ser Ala Se #r Val Ala Ile Gly Glu            340       #           345       #           350Ser Gly Arg Leu Ser Ser Gly Val Glu Gly Se #r Asn Val Arg Ala Leu        355           #       360           #       365Asn Glu Ala Gln Asn Leu Arg Ala Thr Thr Se #r Ser Gly Ser Val Ala    370               #   375               #   380Val Arg Lys Glu Glu Lys Lys Lys Leu Ile Gl #y Asn Ala Ala Val Phe385                 3 #90                 3 #95                 4 #00Tyr Gly Asn Tyr Lys Asn Asn Ala Ser Val Th #r Ile Ala Asp His Ala                405   #               410   #               415Glu Leu Val Ser Glu Gly Lys Ile Asp Ile As #n Ser Glu Asn Lys Ile            420       #           425       #           430Glu Tyr Lys Asn Pro Ser Lys Met Ala Lys Se #r Val Ile Asp Lys Leu        435           #       440           #       445Glu Leu Leu Lys Arg Ala Phe Gly Lys Glu Th #r Lys Thr Pro Glu Tyr    450               #   455               #   460Asp Pro Lys Asp Ile Glu Ser Ile Glu Lys Le #u Leu Asn Ala Phe Ser465                 4 #70                 4 #75                 4 #80Glu Lys Leu Asp Gly Lys Pro Glu Leu Leu Le #u Asn Gly Glu Arg Met                485   #               490   #               495Thr Ile Ile Leu Pro Asp Gly Thr Ser Lys Th #r Gly Thr Ala Ile Glu            500       #           505       #           510Ile Ala Asn Tyr Val Gln Gly Glu Met Lys Ly #s Leu Glu Glu Lys Leu        515           #       520           #       525Pro Lys Gly Phe Lys Ala Phe Ser Glu Gly Le #u Ser Gly Leu Ile Lys    530               #   535               #   540Glu Thr Leu Asn Phe Thr Gly Val Gly Asn Ty #r Ala Asn Phe His Thr545                 5 #50                 5 #55                 5 #60Phe Thr Ser Ser Gly Ala Asn Gly Glu Arg As #p Val Ser Ser Val Gly                565   #               570   #               575Gly Ala Val Ser Trp Val Glu Gln Glu Asn Ty #r Ser Lys Val Ser Val            580       #           585       #           590Gly Lys Gly Ala Lys Leu Ala Ala Lys Lys As #p Leu Asn Ile Lys Ala        595           #       600           #       605Ile Asn Lys Ala Glu Thr Val Asn Leu Val Gl #y Asn Ile Gly Leu Ala    610               #   615               #   620Arg Ser Ser Thr Ser Gly Ser Ala Val Gly Gl #y Arg Leu Asn Val Gln625                 6 #30                 6 #35                 6 #40Arg Ser Lys Asn Ser Ala Ile Val Glu Ala Ly #s Glu Lys Ala Glu Leu                645   #               650   #               655Ser Gly Glu Asn Ile Asn Ala Asp Ala Leu As #n Arg Leu Phe His Val            660       #           665       #           670Ala Gly Ser Phe Asn Gly Gly Ser Gly Gly As #n Ala Ile Asn Gly Met        675           #       680           #       685Gly Ser Tyr Ser Gly Gly Ile Ser Lys Ala Ar #g Val Ser Ile Asp Asp    690               #   695               #   700Glu Ala Tyr Leu Lys Ala Asn Lys Lys Ile Al #a Leu Asn Ser Lys Asn705                 7 #10                 7 #15                 7 #20Asp Thr Ser Val Trp Asn Ala Ala Gly Ser Al #a Gly Ile Gly Thr Lys                725   #               730   #               735Asn Ala Ala Val Gly Val Ala Val Ala Val As #n Asp Tyr Asp Ile Ser            740       #           745       #           750Asn Lys Ala Ser Ile Glu Asp Asn Asp Glu Gl #y Gln Ser Lys Tyr Asp        755           #       760           #       765Lys Asn Lys Asp Asp Glu Val Thr Val Thr Al #a Glu Ser Leu Glu Val    770               #   775               #   780Asp Ala Lys Thr Thr Gly Thr Ile Asn Ser Il #e Ser Val Ala Gly Gly785                 7 #90                 7 #95                 8 #00Ile Asn Lys Val Gly Ser Lys Pro Ser Glu Gl #u Lys Pro Lys Ser Glu                805   #               810   #               815Glu Arg Pro Glu Gly Phe Phe Gly Lys Ile Gl #y Asn Lys Val Asp Ser            820       #           825       #           830Val Lys Asn Lys Ile Thr Asp Ser Met Asp Se #r Leu Thr Glu Lys Ile        835           #       840           #       845Thr Asn Tyr Ile Ser Glu Gly Val Lys Lys Al #a Gly Asn Leu Pro Ser    850               #   855               #   860Asn Val Ser His Thr Pro Asp Lys Gly Pro Se #r Phe Ser Leu Gly Ala865                 8 #70                 8 #75                 8 #80Ser Gly Ser Val Ser Phe Asn Asn Ile Lys Ly #s Glu Thr Ser Ala Val                885   #               890   #               895Val Asp Gly Val Lys Ile Asn Leu Lys Gly Al #a Asn Lys Lys Val Glu            900       #           905       #           910Val Thr Ser Ser Asp Ser Thr Phe Val Gly Al #a Trp Gly Gly Ser        915           #       920           #       925<210> SEQ ID NO 4 <211> LENGTH: 714 <212> TYPE: PRT<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 4Gly Ala Ser Gly Ser Val Ser Phe Asn Asn Il #e Lys Lys Glu Thr Ser  1               5  #                 10  #                 15Ala Val Val Asp Gly Val Lys Ile Asn Leu Ly #s Gly Ala Asn Lys Lys             20      #             25      #             30Val Glu Val Thr Ser Ser Asp Ser Thr Phe Va #l Gly Ala Trp Gly Gly         35          #         40          #         45Ser Ala Ala Leu Gln Trp Asn His Ile Gly Se #r Gly Asn Ser Asn Ile     50              #     55              #     60Ser Ala Gly Leu Ala Gly Ala Ala Ala Val As #n Asn Ile Gln Ser Lys 65                  # 70                  # 75                  # 80Thr Ser Ala Leu Val Lys Asn Ser Asp Ile Ar #g Asn Ala Asn Lys Phe                 85  #                 90  #                 95Lys Val Asn Ala Leu Ser Gly Gly Thr Gln Va #l Ala Ala Gly Ala Gly            100       #           105       #           110Leu Glu Ala Val Lys Glu Ser Gly Gly Gln Gl #y Lys Ser Tyr Leu Leu        115           #       120           #       125Gly Thr Ser Ala Ser Ile Asn Leu Val Asn As #n Glu Val Ser Ala Lys    130               #   135               #   140Ser Glu Asn Asn Thr Val Ala Gly Glu Ser Gl #u Ser Gln Lys Met Asp145                 1 #50                 1 #55                 1 #60Val Asp Val Thr Ala Tyr Gln Ala Asp Thr Gl #n Val Thr Gly Ala Leu                165   #               170   #               175Asn Leu Gln Ala Gly Lys Ser Asn Gly Thr Va #l Gly Ala Thr Val Thr            180       #           185       #           190Val Ala Lys Leu Asn Asn Lys Val Asn Ala Se #r Ile Ser Gly Gly Arg        195           #       200           #       205Tyr Thr Asn Val Asn Arg Ala Asp Ala Lys Al #a Leu Leu Ala Thr Thr    210               #   215               #   220Gln Val Thr Ala Ala Val Thr Thr Gly Gly Th #r Ile Ser Ser Gly Ala225                 2 #30                 2 #35                 2 #40Gly Leu Gly Asn Tyr Gln Gly Ala Val Ser Va #l Asn Lys Ile Asp Asn                245   #               250   #               255Asp Val Glu Ala Ser Val Asp Lys Ser Ser Il #e Glu Gly Ala Asn Glu            260       #           265       #           270Ile Asn Val Ile Ala Lys Asp Val Lys Gly Se #r Ser Asp Leu Ala Lys        275           #       280           #       285Glu Tyr Gln Ala Leu Leu Asn Gly Lys Asp Ly #s Lys Tyr Leu Glu Asp    290               #   295               #   300Arg Gly Ile Asn Thr Thr Gly Asn Gly Tyr Ty #r Thr Lys Glu Gln Leu305                 3 #10                 3 #15                 3 #20Glu Lys Ala Lys Lys Lys Glu Gly Ala Val Il #e Val Asn Ala Ala Leu                325   #               330   #               335Ser Val Ala Gly Thr Asp Lys Ser Ala Gly Gl #y Val Ala Ile Ala Val            340       #           345       #           350Asn Thr Val Lys Asn Lys Phe Lys Ala Glu Le #u Ser Gly Ser Asn Lys        355           #       360           #       365Glu Ala Gly Glu Asp Lys Ile His Ala Lys Hi #s Val Asn Val Glu Ala    370               #   375               #   380Lys Ser Ser Thr Val Val Val Asn Ala Ala Se #r Gly Leu Ala Ile Ser385                 3 #90                 3 #95                 4 #00Lys Asp Ala Phe Ser Gly Met Gly Ser Gly Al #a Trp Gln Asp Leu Ser                405   #               410   #               415Asn Asp Thr Ile Ala Lys Val Asp Lys Gly Ar #g Ile Ser Ala Asp Ser            420       #           425       #           430Leu Asn Val Asn Ala Asn Asn Ser Ile Leu Gl #y Val Asn Val Ala Gly        435           #       440           #       445Thr Ile Ala Gly Ser Leu Ser Thr Ala Val Gl #y Ala Ala Phe Ala Asn    450               #   455               #   460Asn Thr Leu His Asn Lys Thr Ser Ala Leu Il #e Thr Gly Thr Lys Val465                 4 #70                 4 #75                 4 #80Asn Pro Phe Ser Gly Lys Asn Thr Lys Val As #n Val Gln Ala Leu Asn                485   #               490   #               495Asp Ser His Ile Thr Asn Val Ser Ala Gly Gl #y Ala Ala Ser Ile Lys            500       #           505       #           510Gln Ala Gly Ile Gly Gly Met Val Ser Val As #n Arg Gly Ser Asp Glu        515           #       520           #       525Thr Glu Ala Leu Val Ser Asp Ser Glu Phe Gl #u Gly Val Ser Ser Phe    530               #   535               #   540Asn Val Asp Ala Lys Asp Gln Lys Thr Ile As #n Thr Ile Ala Gly Asn545                 5 #50                 5 #55                 5 #60Ala Asn Gly Gly Lys Ala Ala Gly Val Gly Al #a Thr Val Ala His Thr                565   #               570   #               575Asn Ile Gly Lys Gln Ser Val Ile Ala Ile Va #l Lys Asn Ser Lys Ile            580       #           585       #           590Thr Thr Ala Asn Asp Gln Asp Arg Lys Asn Il #e Asn Val Thr Ala Lys        595           #       600           #       605Asp Tyr Thr Met Thr Asn Thr Ile Ala Val Gl #y Val Gly Gly Ala Lys    610               #   615               #   620Gly Ala Ser Val Gln Gly Ala Ser Ala Ser Th #r Thr Leu Asn Lys Thr625                 6 #30                 6 #35                 6 #40Val Ser Ser His Val Asp Gln Thr Asp Ile As #p Lys Asp Leu Glu Glu                645   #               650   #               655Glu Asn Asn Gly Asn Lys Glu Lys Ala Asn Va #l Asn Val Leu Ala Glu            660       #           665       #           670Asn Thr Ser Gln Val Val Thr Asn Ala Thr Va #l Leu Ser Gly Ala Ser        675           #       680           #       685Gly Gln Ala Ala Val Gly Ala Gly Val Ala Va #l Asn Lys Ile Thr Gln    690               #   695               #   700Asn Thr Ser Ala His Ile Lys Asn Ser Thr 705                 7 #10<210> SEQ ID NO 5 <211> LENGTH: 628 <212> TYPE: PRT<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 5Ala Val Gly Ala Gly Val Ala Val Asn Lys Il #e Thr Gln Asn Thr Ser  1               5  #                 10  #                 15Ala His Ile Lys Asn Ser Thr Gln Asn Val Ar #g Asn Ala Leu Val Lys             20      #             25      #             30Ser Lys Ser His Ser Ser Ile Lys Thr Ile Gl #y Ile Gly Ala Gly Val         35          #         40          #         45Gly Ala Gly Gly Ala Gly Val Thr Gly Ser Va #l Ala Val Asn Lys Ile     50              #     55              #     60Val Asn Asn Thr Ile Ala Glu Leu Asn His Al #a Lys Ile Thr Ala Lys 65                  # 70                  # 75                  # 80Gly Asn Val Gly Val Ile Thr Glu Ser Asp Al #a Val Ile Ala Asn Tyr                 85  #                 90  #                 95Ala Gly Thr Val Ser Gly Val Ala Arg Ala Al #a Ile Gly Ala Ser Thr            100       #           105       #           110Ser Val Asn Glu Ile Thr Gly Ser Thr Lys Al #a Tyr Val Lys Asp Ser        115           #       120           #       125Thr Val Ile Ala Lys Glu Glu Thr Asp Asp Ty #r Ile Thr Thr Gln Gly    130               #   135               #   140Gln Val Asp Lys Val Val Asp Lys Val Phe Ly #s Asn Leu Asn Ile Asn145                 1 #50                 1 #55                 1 #60Glu Asp Leu Ser Gln Lys Arg Lys Ile Ser As #n Lys Lys Gly Phe Val                165   #               170   #               175Thr Asn Ser Ser Ala Thr His Thr Leu Lys Se #r Leu Leu Ala Asn Ala            180       #           185       #           190Ala Gly Ser Gly Gln Ala Gly Val Ala Gly Th #r Val Asn Ile Asn Lys        195           #       200           #       205Val Tyr Gly Glu Thr Glu Ala Leu Val Glu As #n Ser Ile Leu Asn Ala    210               #   215               #   220Lys His Tyr Ser Val Lys Ser Gly Asp Tyr Th #r Asn Ser Ile Gly Val225                 2 #30                 2 #35                 2 #40Val Gly Ser Val Gly Val Gly Gly Asn Val Gl #y Val Gly Ala Ser Ser                245   #               250   #               255Asp Thr Asn Ile Ile Lys Arg Asn Thr Lys Th #r Arg Val Gly Lys Thr            260       #           265       #           270Thr Met Ser Asp Glu Gly Phe Gly Glu Glu Al #a Glu Ile Thr Ala Asp        275           #       280           #       285Ser Lys Gln Gly Ile Ser Ser Phe Gly Val Gl #y Val Ala Ala Ala Gly    290               #   295               #   300Val Gly Ala Gly Val Ala Gly Thr Val Ser Va #l Asn Gln Phe Ala Gly305                 3 #10                 3 #15                 3 #20Lys Thr Glu Val Asp Val Glu Glu Ala Lys Il #e Leu Val Lys Lys Ala                325   #               330   #               335Glu Ile Thr Ala Lys Arg Tyr Ser Ser Val Al #a Ile Gly Asn Ala Ala            340       #           345       #           350Val Gly Val Ala Ala Lys Gly Ala Gly Ile Gl #y Ala Ala Val Ala Val        355           #       360           #       365Thr Lys Asp Glu Ser Asn Thr Arg Ala Arg Va #l Lys Asn Ser Lys Ile    370               #   375               #   380Met Thr Arg Asn Lys Leu Asp Val Ile Ala Gl #u Asn Glu Ile Lys Ser385                 3 #90                 3 #95                 4 #00Gly Thr Gly Ile Gly Ser Ala Gly Ala Gly Il #e Leu Ala Ala Gly Val                405   #               410   #               415Ser Gly Val Val Ser Val Asn Asn Ile Ala As #n Lys Val Glu Thr Asp            420       #           425       #           430Ile Asp His Ser Thr Leu His Ser Ser Thr As #p Val Asn Val Lys Ala        435           #       440           #       445Leu Asn Lys Ile Ser Asn Ser Leu Thr Ala Gl #y Gly Gly Ala Ala Gly    450               #   455               #   460Leu Ala Ala Val Thr Gly Val Val Ser Val As #n Thr Ile Asn Ser Ser465                 4 #70                 4 #75                 4 #80Val Ile Ala Arg Val His Asn Asn Ser Asp Le #u Thr Ser Val Arg Glu                485   #               490   #               495Lys Val Asn Val Thr Ala Lys Glu Glu Lys As #n Ile Lys Gln Thr Ala            500       #           505       #           510Ala Asn Ala Gly Ile Gly Gly Ala Ala Ile Gl #y Ala Asn Val Leu Val        515           #       520           #       525Asn Asn Phe Gly Thr Ala Val Glu Asp Arg Ly #s Asn Ser Glu Gly Lys    530               #   535               #   540Gly Thr Glu Val Leu Lys Thr Leu Asp Glu Va #l Asn Lys Glu Gln Asp545                 5 #50                 5 #55                 5 #60Lys Lys Val Asn Asp Ala Thr Lys Lys Ile Le #u Gln Ser Ala Gly Ile                565   #               570   #               575Ser Thr Glu Asp Thr Ser Val Lys Ala Asp Ar #g Gly Asp Thr Gln Gly            580       #           585       #           590Glu Gly Ile Lys Ala Ile Val Lys Thr Ser As #p Ile Ile Gly Lys Asn        595           #       600           #       605Val Asp Ile Thr Thr Glu Asp Lys Asn Asn Il #e Thr Ser Thr Gly Gly    610               #   615               #   620 Leu Gly Thr Ala 625<210> SEQ ID NO 6 <211> LENGTH: 773 <212> TYPE: PRT<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 6Gly Ile Lys Ala Ile Val Lys Thr Ser Asp Il #e Ile Gly Lys Asn Val  1               5  #                 10  #                 15Asp Ile Thr Thr Glu Asp Lys Asn Asn Ile Th #r Ser Thr Gly Gly Leu             20      #             25      #             30Gly Thr Ala Gly Leu Ala Ser Ala Ser Gly Th #r Val Ala Val Thr Asn         35          #         40          #         45Ile Lys Arg Asn Ser Gly Val Thr Val Glu As #n Ser Phe Val Lys Ala     50              #     55              #     60Ala Glu Lys Val Asn Val Arg Ser Asp Ile Th #r Gly Asn Val Ala Leu 65                  # 70                  # 75                  # 80Thr Ala Tyr Gln Gly Pro Val Gly Ala Leu Gl #y Ile Gly Ala Ala Tyr                 85  #                 90  #                 95Ala Glu Leu Asn Ser Asn Gly Arg Ser Asn Il #e Ser Ile Lys Asn Ser            100       #           105       #           110Lys Leu Leu Gly Lys Asn Ile Asp Val Ile Va #l Lys Asp Lys Ser Glu        115           #       120           #       125Leu Arg Ala Glu Ala Lys Gly Leu Thr Val Gl #y Ala Val Ala Ala Gly    130               #   135               #   140Ala Ile Ile Ser Lys Ala Lys Asn Glu Met As #n Ser Glu Val Glu Ile145                 1 #50                 1 #55                 1 #60Glu Lys Ser Ile Phe Asn Glu Glu Asn Arg Va #l Thr Ser Pro Ser Lys                165   #               170   #               175Gly Ile Gly Arg Glu Ile Asn Val Lys Val Gl #u Lys Glu Asn Arg Val            180       #           185       #           190Thr Ala Glu Ser Gln Gly Ala Ser Val Gly Al #a Val Ala Gly Ala Gly        195           #       200           #       205Ile Ile Ser Glu Ala Lys Asp Ala Gly Ser Se #r Tyr Leu Lys Val Ser    210               #   215               #   220Thr Lys Ser Gly Arg Ser Ile Phe His Ala As #p Asn Val Asn Met Glu225                 2 #30                 2 #35                 2 #40Ala Thr His Lys Met Lys Val Thr Ala Val Se #r Lys Ala Val Thr Gly                245   #               250   #               255Ser Val Leu Gly Gly Val Gly Val Thr Lys Al #a Glu Ala Thr Ala Ala            260       #           265       #           270Gly Lys Thr Met Val Glu Val Glu Glu Gly As #n Leu Phe Arg Thr Asn        275           #       280           #       285Arg Leu Asn Ala Ile Ser Lys Val Glu Gly Le #u Asp Glu Asp Lys Val    290               #   295               #   300Thr Ala Lys Ser Ser Val Val Ser Gly Asn Gl #y Gly Gly Ile Ala Gly305                 3 #10                 3 #15                 3 #20Ala Gly Val Asn Thr Ser Thr Ala Gln Ser As #n Thr Glu Ser Val Val                325   #               330   #               335Arg Leu Arg Lys Gln Asp Tyr Glu Asn Asn As #p Tyr Thr Lys Lys Tyr            340       #           345       #           350Ile Ser Glu Val Asn Ala Leu Ala Leu Asn As #p Thr Lys Asn Glu Ala        355           #       360           #       365Asn Ile Glu Ser Leu Ala Val Ala Gly Val Hi #s Ala Gln Gly Thr Asn    370               #   375               #   380Lys Ala Phe Thr Arg Ser Asn Lys Leu Thr Se #r Thr Thr Val Asn Gly385                 3 #90                 3 #95                 4 #00Gly Asn Val Ser Gln Leu Arg Ala Lys Ala Le #u Ala Lys Asn Glu Asn                405   #               410   #               415Tyr Gly Asn Val Lys Gly Thr Gly Gly Ala Le #u Val Gly Ala Glu Thr            420       #           425       #           430Ala Ala Val Glu Asn Tyr Thr Lys Ser Thr Th #r Gly Ala Leu Val Ala        435           #       440           #       445Gly Asn Trp Glu Ile Gly Asp Lys Leu Glu Th #r Ile Ala Arg Asp Asn    450               #   455               #   460Thr Ile Val Arg Val Asn Gly Asp Gly Thr Ly #s Gly Gly Leu Val Gly465                 4 #70                 4 #75                 4 #80Lys Asn Gly Ile Ser Val Lys Asn Thr Ile Se #r Gly Glu Thr Lys Ser                485   #               490   #               495Ser Ile Glu Asp Lys Ala Arg Ile Val Gly Th #r Gly Ser Val Asn Val            500       #           505       #           510Asp Ala Leu Asn Glu Leu Asp Val Asp Leu Gl #n Gly Lys Ser Gly Gly        515           #       520           #       525Tyr Gly Gly Ile Gly Ile Gly Asn Val Asp Va #l Asn Asn Val Ile Lys    530               #   535               #   540Lys Asn Val Glu Ala Lys Ile Gly Arg His Al #a Ile Val Glu Thr Thr545                 5 #50                 5 #55                 5 #60Gly Lys Gln Glu Tyr Gln Ala Phe Thr Arg Al #a Lys Val Asn Ile Leu                565   #               570   #               575Gly Lys Gly Asp Ala Ala Ala Ala Ala Ala Il #e Ser Asn Val His Ile            580       #           585       #           590Ser Asn Glu Met Asp Ile Lys Asn Leu Ala Ly #s Gln Tyr Ala Ser Ser        595           #       600           #       605Gln Leu Ile Thr Lys Asn Ser Lys Asn Asn Il #e Thr Leu Ala Ser Ser    610               #   615               #   620Ser Glu Ser Asn Val Asn Val His Gly Val Al #a Glu Ala Arg Gly Ala625                 6 #30                 6 #35                 6 #40Gly Ala Lys Ala Thr Val Ser Val Lys Asn Gl #n Ile Asn Arg Thr Asn                645   #               650   #               655Asn Val Asp Leu Ala Gly Lys Ile Lys Thr Gl #u Gly Asn Ile Asn Val            660       #           665       #           670Tyr Ala Gly Tyr Asp Lys Asn Tyr Asn Ile Se #r Lys Thr Asn Ser Lys        675           #       680           #       685Ala Ile Ala Asp Ala Lys Ser His Ala Ala Al #a Ala Ser Ala Thr Ala    690               #   695               #   700Thr Ile Glu Lys Asn Glu Val Lys Phe Asn As #n Ala Ile Arg Glu Phe705                 7 #10                 7 #15                 7 #20Lys Asn Asn Leu Ala Arg Leu Glu Gly Lys Al #a Asn Lys Lys Thr Ser                725   #               730   #               735Val Gly Ser Asn Gln Val Asp Trp Tyr Thr As #p Lys Tyr Thr Trp His            740       #           745       #           750Ser Ser Glu Lys Ala Tyr Lys Lys Leu Thr Ty #r Gln Ser Lys Arg Gly        755           #       760           #       765Glu Lys Gly Lys Lys     770 <210> SEQ ID NO 7 <211> LENGTH: 338<212> TYPE: PRT <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 7 Ile Asn Met Ala Ser Gly Lys Val Pro Gly Th#r Thr Asp Tyr Phe Val   1               5  #                 10 #                 15 Gln Ile Tyr Glu Pro Lys Arg Gln Gln Phe Ph#e Val Phe Ala Asp Asn              20      #             25     #             30 Leu Gly Gln Lys Asn Thr Gly Glu Leu Arg Tr#p Gly Leu Asn Tyr Ile          35          #         40         #         45 Asn Asn Ser Val Thr Gly Asn Arg Asp Gln Le#u Ser Leu Thr Ser Leu      50              #     55             #     60 Val Thr Glu Gly Thr Ala Ser Leu Ser Ser Ph#e Tyr Thr Phe Pro Val  65                  # 70                 # 75                  # 80 Ser Lys Lys Gly Thr Lys Ile Ser Leu Gln Hi#s Ser Val Gly Lys Leu                  85  #                 90 #                 95 Lys His Ile Gln Gly Ala Leu Lys His Lys Il#e Thr Gly Asn Ser Tyr             100       #           105      #           110 Ser Tyr Gly Val Gly Ile Val His Pro Ile Le#u Val His Glu Lys Asn         115           #       120          #       125 Lys Val Glu Leu Ser Leu Asp Trp Val Lys Gl#n Arg Thr Val Thr Asp     130               #   135              #   140 Leu Leu Lys Leu Lys Trp Val Asn Asn Arg Le#u Ser Lys Tyr Thr Ala 145                 1 #50                 1#55                 1 #60 Gly Ile Gly Ile Ser His Tyr Glu Glu Asp Se#r Val Phe Tyr Thr Lys                 165   #               170  #               175 Gln Asn Ile Thr Lys Gly Lys Phe Ile Pro Il#e Ser Gly Asp Ala Arg             180       #           185      #           190 Asn Tyr Thr Lys Tyr Asp Met Phe Leu Ile Ty#r Gln Lys Asn Leu Lys         195           #       200          #       205 Tyr Asn Thr Leu Val Thr Leu Lys Met Ala Gl#y Gln Tyr Ser Leu Ser     210               #   215              #   220 Lys Lys Leu Pro Ser Val Glu Gln Ile Tyr Al#a Gly Gly Ala Tyr Asn 225                 2 #30                 2#35                 2 #40 Val Arg Gly Tyr Pro Glu Asn Phe Met Gly Al#a Glu His Gly Val Phe                 245   #               250  #               255 Phe Asn Ala Glu Leu Ser Lys Leu Val Glu As#n Lys Gly Glu Phe Phe             260       #           265      #           270 Val Phe Leu Asp Gly Ala Ser Leu His Gly Gl#u Ser Ala Trp Gln Glu         275           #       280          #       285 Asn Arg Ile Phe Ser Ser Gly Phe Gly Tyr Ly#s Ile Arg Phe Leu Glu     290               #   295              #   300 Lys Asn Asn Ile Ala Val Ser Met Ala Phe Pr#o Trp Lys Lys Lys Ile 305                 3 #10                 3#15                 3 #20 Asn Ser Ile Ser Val Asp Ser Asn Arg Ile Ty#r Ile Thr Ile Asn His                 325   #               330  #               335 Glu Phe <210> SEQ ID NO 8 <211> LENGTH: 9726<212> TYPE: DNA <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 8atgagcggca tcaaaaataa cgttcagagg acaaggaaga ggatatcaga tt#ctaaaaaa     60gttttaatga ttttgggatt gttgattaac actatgacgg tgagggctaa tg#atacaatc    120accgcgactg agaattttgg aacaaaaata gaaaaaaagg ataatgttta tg#acattact    180acaaacaaga ttcaagggga gaacgctttt aacagtttta atagatttgc tt#taacagaa    240aataatatag caaatctata ttttggggaa aagaatagta cgggggtaaa ta#atcttttt    300aactttgtca atggaaaaat tgaagtagat gggattatca acggaattcg ag#aaaataaa    360attggaggaa atttatattt cttaagctcg gaagggatgg cagtaggaaa aa#atggagtt    420atcaatgctg gttcttttca ttctattatt ccaaaacaag atgattttaa ga#aggctttg    480gaagaagcca aacatggtaa agtttttaat ggaatcattc cagtagatgg aa#aagtaaaa    540attccattga atccgaatgg aagcattacg gtagaaggaa aaatcaatgc tg#ttgaaggc    600atcggtttat atgcggcgga tattagattg aaagatactg caatactaaa ga#caggaatt    660acagatttta aaaatttagt caatattagt gatcgaataa attctggtct ga#ccggagat    720ttaaaagcta ccaagacaaa atctggagat attattcttt cagctcacat ag#attctcct    780caaaaagcta tgggaaaaaa ttcaactgtt ggaaagagaa tagaagaata tg#taaaagga    840aataccaaag caaatattga atctgatgct gtattggaag cagatggaaa ta#taaaaatt    900agtgcgaaag ctacaaatgg gagatttata aagaaagaag gggaaaaaga aa#cttataac    960actcctttaa gtttatcaga tgtggaagct tccgtaagag taaataaagg aa#aagtcata   1020ggaaagaatg ttgacattac agctgaagca aagaatttct atgatgcaac tt#tagttact   1080aagcttgcaa agcactcttt tagctttgtt acaggttcta tttctcctat ca#atttaaat   1140ggatttttag gtttattgac aagtaagtcc agtgtcgtta ttggaaaaga tg#ccaaagtc   1200gaagcaacag aaggaaaggc aaatattcat tcttacagtg gagtaagagc aa#ctatggga   1260gcagctactt ctccattaaa aattaccaat ttatatttgg agaaagccaa tg#gaaaactt   1320ctcagtatcg gagcgggata tatttctgca aaaagtaatt ccaatgtaac ta#ttgaagga   1380gaagtaaaat cgaagggaag agcagatatt acttcaaaat ctgaaaatac ta#ttgatgct   1440tctgtttctg ttggaacgat gagagattcc aataaagtag ctctttcagt at#tggtgacg   1500gaaggagaaa ataaatcttc cgtcaagatt gctaaaggag caaaagtaga at#cagaaacg   1560gatgatgtaa atgtgagaag tgaagcgatt aattccattc gagctgctgt aa#aaggtgga   1620ttgggggata gtggtaatgg ggttgtggct gcaaatattt ctaactataa tg#cttcctcc   1680cgtatagatg tagatggata tctacatgcc aagaagcgac taaatgtgga gg#ctcataac   1740attactaaaa atagtgttct gcaaacagga tctgatttgg gaacttccaa gt#ttatgaat   1800gatcacgttt atgaatcagg tcatctaaaa tcaattttag atgcaataaa ac#agcggttt   1860ggaggagaca gtgtcaatga ggaaataaag aataagctaa cgaacttatt ta#gtgtcggt   1920gtgtctgcaa ccatagcaaa tcataataat tctgcttctg tggcaatagg ag#agagtgga   1980agactttctt caggagtgga agggagtaat gtaagggcat taaatgaagc tc#aaaatctt   2040cgagcgacta cgtcaagtgg aagtgtggct gtacgaaagg aagaaaaaaa ga#aacttatt   2100ggaaatgcag cagtttttta tggaaactat aaaaataatg cttctgtgac aa#ttgccgat   2160catgctgaat tggtatcgga aggaaaaatt gatatcaaca gtgaaaataa aa#ttgaatat   2220aaaaatcctt caaaaatggc aaagtctgtt attgataaat tagaactttt aa#agagagct   2280tttggaaaag aaacgaaaac tccagaatat gatccgaaag atattgaatc ta#ttgaaaaa   2340ttattgaatg cattttcaga aaaattggat ggaaaaccgg agcttttact aa#atggtgaa   2400agaatgacaa ttattcttcc ggatggaact tcaaaaacag gaactgctat ag#aaattgca   2460aactatgttc agggagaaat gaaaaaatta gaggaaaaat taccgaaagg at#ttaaagct   2520ttttcagaag gattgagtgg actgattaaa gaaactttga attttacagg ag#taggaaat   2580tatgcaaatt ttcacacttt tacctcttcc ggagctaatg gagaaagaga tg#tttcttct   2640gtgggaggag ctgtttcgtg ggtagaacag gagaattata gcaaggtatc cg#ttggaaaa   2700ggagctaaac ttgctgcaaa aaaagattta aatataaaag ctatcaataa ag#cagaaaca   2760gtgaatttag ttggaaatat tggacttgcg agaagcagta catccggaag tg#cagtcgga   2820ggaagattaa atgttcaaag atcgaaaaat tcagctatcg tagaagctaa ag#aaaaagct   2880gaattatcag gagaaaatat taatgcagat gcattgaaca gactttttca tg#tagcggga   2940tcttttaatg gtggctcagg tgggaatgca atcaatggaa tgggaagtta ta#gtggaggt   3000atcagtaagg caagagtttc cattgatgac gaagcatatt tgaaagctaa ta#aaaaaatt   3060gctttaaaca gtaagaatga tacttctgtt tggaatgctg ccggttcagc gg#gaatcgga   3120acgaaaaatg cggcggtcgg ggttgctgtt gcggtaaatg attatgatat tt#caaacaaa   3180gcttccattg aagataatga cgaaggacaa agtaaatatg ataagaataa ag#atgatgaa   3240gtaacagtaa ctgcggaatc tttagaagta gatgcaaaaa cgaccggaac aa#tcaacagt   3300atttctgttg ccggaggaat taataaggtt ggaagtaaac cgagtgaaga aa#aaccgaaa   3360tcagaagaaa gaccagaggg attttttggc aaaatcggaa acaaagtgga ct#ctgtaaaa   3420aataaaatta cggatagtat ggattcatta acagaaaaaa ttacaaatta ca#tttctgaa   3480ggagtaaaaa aagcggggaa tcttccttcg aacgtttctc atactcccga ta#aaggaccg   3540tctttcagtt tgggagcttc tggaagtgtt tctttcaata atattaaaaa gg#aaacatct   3600gctgtcgtag atggagtaaa gataaatttg aagggagcaa ataaaaaggt ag#aggtgact   3660tcttctgatt ctacttttgt tggagcatgg ggcggatctg ctgcacttca gt#ggaatcat   3720attggaagtg gaaatagcaa catcagtgct ggtttagctg gagcggctgc tg#taaataat   3780attcaaagta aaacaagtgc tttggttaaa aatagtgata ttcgaaatgc ca#ataaattt   3840aaagtaaatg ctttgagtgg aggaactcaa gtagcagcag gagcaggttt gg#aagcagtt   3900aaagaaagtg gaggacaagg aaaaagttat ctattgggaa cttctgcttc ta#tcaactta   3960gtgaacaatg aagtttctgc aaaatcagaa aataatacag tagcaggaga at#ctgaaagc   4020caaaaaatgg atgttgatgt cactgcttat caagcggaca cccaagtgac ag#gagcttta   4080aatttacaag ctggaaagtc aaatggaact gtaggggcta ctgtgactgt tg#ccaaatta   4140aacaacaaag taaatgcttc tattagtggt gggagatata ctaacgttaa tc#gagcggac   4200gcaaaagctc ttttagcaac cactcaagtg actgctgcag tgacgacggg ag#ggacaatt   4260agttctggag cgggattagg aaattatcaa ggggctgttt ctgtcaataa ga#ttgacaat   4320gacgtggaag ctagcgttga taaatcttcc atcgaaggag ctaatgaaat ca#atgtcatt   4380gccaaagatg tcaaaggaag ttctgatcta gcaaaagaat atcaggcttt ac#taaatgga   4440aaagataaaa aatatttaga agatcgtggt attaatacga ctggaaatgg tt#attatacg   4500aaggaacaac tagaaaaagc aaagaaaaaa gaaggagcgg tcattgtaaa tg#ctgcttta   4560tcggttgctg gaacggataa atccgctgga ggagtagcta ttgcagtcaa ta#ctgttaaa   4620aataaattta aagcagaatt gagtggaagc aataaggaag ccggagagga ta#aaattcat   4680gcgaaacatg taaatgtgga ggcaaaatca tctactgttg ttgtgaatgc gg#cttctgga   4740cttgctatca gcaaagatgc tttttcagga atgggatctg gagcatggca ag#acttatca   4800aatgacacga ttgcaaaggt ggataaagga agaatttctg ctgattcctt aa#atgtgaac   4860gcaaataatt ccattcttgg ggtgaatgtt gcgggaacca ttgccggttc tc#tttctacg   4920gcggtaggag ctgcttttgc gaataatact cttcataata aaacctctgc tt#tgattaca   4980ggaacgaagg taaatccttt tagtggaaag aatacaaaag tcaatgtaca ag#ctttgaat   5040gattctcata ttacaaacgt ttctgctgga ggcgctgcaa gtattaagca gg#ctggaatc   5100ggaggaatgg tatctgtcaa tcgtggttct gatgaaacgg aagctttagt ta#gtgattct   5160gagtttgaag gagtaagttc tttcaatgta gatgcaaaag atcaaaaaac aa#taaataca   5220attgccggaa atgcaaatgg aggaaaagcg gctggagttg gagcaacagt tg#ctcataca   5280aatattggaa aacaatcagt tatagctatt gtaaaaaaca gtaaaattac aa#cggcgaat   5340gatcaagata gaaaaaatat caatgtgact gcaaaagatt atactatgac ca#atactata   5400gcagtcggag ttggaggagc aaaaggagcc tctgtgcaag gagcttctgc aa#gtactacc   5460ttgaataaga cagtttcttc tcatgttgat caaactgata ttgacaaaga tt#tagaggaa   5520gaaaataatg gaaataagga aaaggcaaat gttaatgttc tagctgaaaa ta#cgagtcaa   5580gtggtcacaa atgcgacagt gctttccgga gcaagtggac aagctgcagt ag#gagctgga   5640gtagcagtta ataaaattac acaaaatact tctgcacata taaaaaatag ta#ctcaaaat   5700gtacgaaatg ctttggtaaa aagcaaatct cattcatcta ttaaaacaat tg#gaattgga   5760gctggagttg gagctggagg agctggagtg acaggttctg tagcagtgaa ta#agattgta   5820aataatacga tagcagaatt aaatcatgca aaaatcactg cgaagggaaa tg#tcggagtt   5880attacagagt ctgatgcggt aattgctaat tatgcaggaa cagtgtctgg ag#tggcccgt   5940gcagcaatag gagcctcaac cagtgtgaat gaaattacag gatctacaaa ag#catatgta   6000aaagattcta cagtgattgc taaagaagaa acagatgatt atattactac tc#aagggcaa   6060gtagataaag tggtagataa agtattcaaa aatcttaata ttaacgaaga ct#tatcacaa   6120aaaagaaaaa taagtaataa aaaaggattt gttaccaata gttcagctac tc#atacttta   6180aaatctttat tggcaaatgc cgctggttca ggacaagccg gagtggcagg aa#ctgttaat   6240atcaacaagg tttatggaga aacagaagct cttgtagaaa attctatatt aa#atgcaaaa   6300cattattctg taaaatcagg agattacacg aattcaatcg gagtagtagg tt#ctgttggt   6360gttggtggaa atgtaggagt aggagcttct tctgatacca atattataaa aa#gaaatacc   6420aagacaagag ttggaaaaac tacaatgtct gatgaaggtt tcggagaaga ag#ctgaaatt   6480acagcagatt ctaagcaagg aatttcctct tttggagtcg gagtcgcagc ag#ccggggta   6540ggagccggag tggcaggaac cgtttccgta aatcaatttg caggaaagac gg#aagtagat   6600gtggaagaag caaagatttt ggtaaaaaaa gctgagatta cagcaaaacg tt#atagttct   6660gttgcaattg gaaatgccgc agtcggagtg gctgcaaaag gagctggaat tg#gagcagca   6720gtggcagtta ccaaagatga atcaaacacg agagcaagag tgaaaaattc ta#aaattatg   6780actcgaaaca agttagatgt aatagcagaa aatgagataa aatcaggtac tg#gaatcggt   6840tcagccggag ctggaattct tgcagccgga gtatctggag tggtttctgt ca#ataatatt   6900gcaaataagg tagaaacaga tatcgatcat agtactttac actcttctac tg#atgtaaat   6960gtaaaagctc ttaataaaat ttcgaattcc ttgacagccg gtggaggagc cg#caggtctt   7020gcagcagtta ccggagtggt ttctgttaac actataaata gttctgtgat ag#ctcgagtt   7080cacaataact ctgatttgac ttccgtacga gaaaaagtaa atgtaacggc aa#aagaggaa   7140aaaaatatta agcaaacagc agcaaatgca ggaatcggag gagcagcaat cg#gagccaat   7200gtcttggtaa ataattttgg aacagctgta gaagatagaa aaaattctga ag#gaaaagga   7260acagaagttt taaaaacttt agacgaagtt aacaaagaac aagataaaaa ag#taaatgat   7320gctacgaaaa aaatcttaca atcagcaggt atttctacag aagatacttc tg#taaaagcg   7380gatagaggag atactcaggg agaaggaatt aaagccattg tgaagacttc tg#atattatt   7440ggaaaaaatg tagatattac aacagaggac aagaataata tcacttctac tg#gtggtttg   7500ggaactgcag gtcttgcttc cgcatcagga acagtggcag ttacaaatat ta#aaagaaat   7560tccggagtta ctgttgaaaa ttcttttgtg aaagcagctg aaaaagtaaa tg#ttagatcg   7620gatattacag gaaatgttgc tttaacagca tatcaaggtc ctgtaggagc at#tgggaata   7680ggagctgcct atgcagaatt aaattctaat ggaagatcaa atatcagtat ta#aaaattct   7740aagctattag gaaaaaatat tgatgttatt gtaaaagata aatcggaatt ga#gagcggaa   7800gcaaaaggat taaccgtagg agcggtagct gccggagcca ttatctcaaa ag#caaagaat   7860gaaatgaatt cagaggttga aattgagaag agtattttca atgaagaaaa ta#gagtaact   7920agcccttcta aaggaattgg aagagaaatc aatgtcaaag tggaaaaaga aa#acagagtg   7980actgctgaat ctcaaggagc ttctgtagga gcagtagcag gggcaggaat ta#tttccgaa   8040gcaaaagatg ccggaagctc ttatttgaaa gttagtacaa aatccggaag aa#gtattttt   8100catgcagata atgtgaatat ggaagcaaca cataaaatga aagtaacagc ag#tttctaaa   8160gcagtaacag gttctgtatt gggaggagtt ggagtcacca aggcagaagc ta#ctgctgca   8220ggtaaaacta tggtagaagt tgaggaagga aatttgttca gaacaaatcg at#tgaatgca   8280atttctaaag tagaaggttt ggatgaagat aaagtaactg ctaaatcttc tg#tagtatca   8340ggaaatggag gaggaattgc cggagcagga gtgaatactt ctacagcaca aa#gtaatact   8400gaatccgtag ttcgtttacg aaagcaagat tatgaaaata atgattacac aa#aaaaatat   8460atttcagaag tcaatgctct tgctttaaat gatacaaaga atgaagcgaa ta#tagaatct   8520ttagcggtag ccggtgtgca tgcacaagga acaaacaaag catttacgag at#caaacaag   8580ttaacttcta caactgtaaa tggaggaaac gtatctcaac ttcgtgcaaa ag#ctttggct   8640aaaaatgaaa attatggaaa tgtaaaagga actggaggag ccttagtcgg ag#cggaaaca   8700gcagccgttg aaaattatac aaagagtact acaggagcat tggttgcagg aa#attgggaa   8760attggagata aattagaaac gattgcaaga gataatacga ttgtaagagt ca#acggagac   8820ggaaccaaag gaggtcttgt cggaaagaat ggtatttctg tgaaaaatac aa#tttcaggg   8880gaaacaaaat catccattga agataaagcc agaattgttg gaaccggaag tg#taaatgta   8940gatgctttga atgaacttga tgtagatcta caaggaaaaa gtggtggcta tg#gtggaatt   9000ggtattggaa atgttgatgt aaataatgtg attaagaaaa atgtagaagc ca#aaatcgga   9060agacatgcta ttgtagaaac tactggaaaa caagaatatc aagcatttac aa#gagcaaaa   9120gtaaatattc ttggaaaagg agacgctgca gctgcagctg caatatcgaa tg#tacacatt   9180tccaatgaga tggatattaa aaatttggca aagcagtatg catcttctca at#taataacc   9240aaaaattcaa aaaataatat tactttagca tcaagtagtg aatcgaatgt ga#atgttcat   9300ggggtggctg aagcaagagg tgcaggagcc aaagcgacag ttagtgtaaa ga#atcaaata   9360aatagaacta ataatgttga tttagcagga aaaattaaaa cagagggaaa ca#tcaatgta   9420tatgccggat atgataaaaa ttataatata agtaagacaa attctaaggc ta#ttgcggat   9480gccaaaagtc atgctgcagc tgcttcggca actgccacta ttgaaaaaaa tg#aagtaaaa   9540tttaataatg cgatccgaga atttaaaaat aatctggcaa gattggaagg ga#aagctaat   9600aaaaaaacgt cggtaggatc taatcaggta gactggtata cggataaata ta#catggcat   9660tcttctgaaa aagcatacaa aaaattgaca tatcaatcaa agagaggaga aa#aagggaaa   9720 aaatga                  #                  #                   #         9726 <210> SEQ ID NO 9 <211> LENGTH: 1130<212> TYPE: DNA <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 9atgagcggca tcaaaaataa cgttcagagg acaaggaaga ggatatcaga tt#ctaaaaaa     60gttttaatga ttttgggatt gttgattaac actatgacgg tgagggctaa tg#atacaatc    120accgcgactg agaattttgg aacaaaaata gaaaaaaagg ataatgttta tg#acattact    180acaaacaaga ttcaagggga gaacgctttt aacagtttta atagatttgc tt#taacagaa    240aataatatag caaatctata ttttggggaa aagaatagta cgggggtaaa ta#atcttttt    300aactttgtca atggaaaaat tgaagtagat gggattatca acggaattcg ag#aaaataaa    360attggaggaa atttatattt cttaagctcg gaagggatgg cagtaggaaa aa#atggagtt    420atcaatgctg gttcttttca ttctattatt ccaaaacaag atgattttaa ga#aggctttg    480gaagaagcca aacatggtaa agtttttaat ggaatcattc cagtagatgg aa#aagtaaaa    540attccattga atccgaatgg aagcattacg gtagaaggaa aaatcaatgc tg#ttgaaggc    600atcggtttat atgcggcgga tattagattg aaagatactg caatactaaa ga#caggaatt    660acagatttta aaaatttagt caatattagt gatcgaataa attctggtct ga#ccggagat    720ttaaaagcta ccaagacaaa atctggagat attattcttt cagctcacat ag#attctcct    780caaaaagcta tgggaaaaaa ttcaactgtt ggaaagagaa tagaagaata tg#taaaagga    840aataccaaag caaatattga atctgatgct gtattggaag cagatggaaa ta#taaaaatt    900agtgcgaaag ctacaaatgg gagatttata aagaaagaag gggaaaaaga aa#cttataac    960actcctttaa gtttatcaga tgtggaagct tccgtaagag taaataaagg aa#aagtcata   1020ggaaagaatg ttgacattac agctgaagca aagaatttct atgatgcaac tt#tagttact   1080aagcttgcaa agcactcttt tagctttgtt acaggttcta tttctcctat  #            1130 <210> SEQ ID NO 10 <211> LENGTH: 2780 <212> TYPE: DNA<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 10gggagattta taaagaaaga aggggaaaaa gaaacttata acactccttt aa#gtttatca     60gatgtggaag cttccgtaag agtaaataaa ggaaaagtca taggaaagaa tg#ttgacatt    120acagctgaag caaagaattt ctatgatgca actttagtta ctaagcttgc aa#agcactct    180tttagctttg ttacaggttc tatttctcct atcaatttaa atggattttt ag#gtttattg    240acaagtaagt ccagtgtcgt tattggaaaa gatgccaaag tcgaagcaac ag#aaggaaag    300gcaaatattc attcttacag tggagtaaga gcaactatgg gagcagctac tt#ctccatta    360aaaattacca atttatattt ggagaaagcc aatggaaaac ttctcagtat cg#gagcggga    420tatatttctg caaaaagtaa ttccaatgta actattgaag gagaagtaaa at#cgaaggga    480agagcagata ttacttcaaa atctgaaaat actattgatg cttctgtttc tg#ttggaacg    540atgagagatt ccaataaagt agctctttca gtattggtga cggaaggaga aa#ataaatct    600tccgtcaaga ttgctaaagg agcaaaagta gaatcagaaa cggatgatgt aa#atgtgaga    660agtgaagcga ttaattccat tcgagctgct gtaaaaggtg gattggggga ta#gtggtaat    720ggggttgtgg ctgcaaatat ttctaactat aatgcttcct cccgtataga tg#tagatgga    780tatctacatg ccaagaagcg actaaatgtg gaggctcata acattactaa aa#atagtgtt    840ctgcaaacag gatctgattt gggaacttcc aagtttatga atgatcacgt tt#atgaatca    900ggtcatctaa aatcaatttt agatgcaata aaacagcggt ttggaggaga ca#gtgtcaat    960gaggaaataa agaataagct aacgaactta tttagtgtcg gtgtgtctgc aa#ccatagca   1020aatcataata attctgcttc tgtggcaata ggagagagtg gaagactttc tt#caggagtg   1080gaagggagta atgtaagggc attaaatgaa gctcaaaatc ttcgagcgac ta#cgtcaagt   1140ggaagtgtgg ctgtacgaaa ggaagaaaaa aagaaactta ttggaaatgc ag#cagttttt   1200tatggaaact ataaaaataa tgcttctgtg acaattgccg atcatgctga at#tggtatcg   1260gaaggaaaaa ttgatatcaa cagtgaaaat aaaattgaat ataaaaatcc tt#caaaaatg   1320gcaaagtctg ttattgataa attagaactt ttaaagagag cttttggaaa ag#aaacgaaa   1380actccagaat atgatccgaa agatattgaa tctattgaaa aattattgaa tg#cattttca   1440gaaaaattgg atggaaaacc ggagctttta ctaaatggtg aaagaatgac aa#ttattctt   1500ccggatggaa cttcaaaaac aggaactgct atagaaattg caaactatgt tc#agggagaa   1560atgaaaaaat tagaggaaaa attaccgaaa ggatttaaag ctttttcaga ag#gattgagt   1620ggactgatta aagaaacttt gaattttaca ggagtaggaa attatgcaaa tt#ttcacact   1680tttacctctt ccggagctaa tggagaaaga gatgtttctt ctgtgggagg ag#ctgtttcg   1740tgggtagaac aggagaatta tagcaaggta tccgttggaa aaggagctaa ac#ttgctgca   1800aaaaaagatt taaatataaa agctatcaat aaagcagaaa cagtgaattt ag#ttggaaat   1860attggacttg cgagaagcag tacatccgga agtgcagtcg gaggaagatt aa#atgttcaa   1920agatcgaaaa attcagctat cgtagaagct aaagaaaaag ctgaattatc ag#gagaaaat   1980attaatgcag atgcattgaa cagacttttt catgtagcgg gatcttttaa tg#gtggctca   2040ggtgggaatg caatcaatgg aatgggaagt tatagtggag gtatcagtaa gg#caagagtt   2100tccattgatg acgaagcata tttgaaagct aataaaaaaa ttgctttaaa ca#gtaagaat   2160gatacttctg tttggaatgc tgccggttca gcgggaatcg gaacgaaaaa tg#cggcggtc   2220ggggttgctg ttgcggtaaa tgattatgat atttcaaaca aagcttccat tg#aagataat   2280gacgaaggac aaagtaaata tgataagaat aaagatgatg aagtaacagt aa#ctgcggaa   2340tctttagaag tagatgcaaa aacgaccgga acaatcaaca gtatttctgt tg#ccggagga   2400attaataagg ttggaagtaa accgagtgaa gaaaaaccga aatcagaaga aa#gaccagag   2460ggattttttg gcaaaatcgg aaacaaagtg gactctgtaa aaaataaaat ta#cggatagt   2520atggattcat taacagaaaa aattacaaat tacatttctg aaggagtaaa aa#aagcgggg   2580aatcttcctt cgaacgtttc tcatactccc gataaaggac cgtctttcag tt#tgggagct   2640tctggaagtg tttctttcaa taatattaaa aaggaaacat ctgctgtcgt ag#atggagta   2700aagataaatt tgaagggagc aaataaaaag gtagaggtga cttcttctga tt#ctactttt   2760 gttggagcat ggggcggatc             #                  #                 278 #0 <210> SEQ ID NO 11 <211> LENGTH: 2141<212> TYPE: DNA <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 11ggagcttctg gaagtgtttc tttcaataat attaaaaagg aaacatctgc tg#tcgtagat     60ggagtaaaga taaatttgaa gggagcaaat aaaaaggtag aggtgacttc tt#ctgattct    120acttttgttg gagcatgggg cggatctgct gcacttcagt ggaatcatat tg#gaagtgga    180aatagcaaca tcagtgctgg tttagctgga gcggctgctg taaataatat tc#aaagtaaa    240acaagtgctt tggttaaaaa tagtgatatt cgaaatgcca ataaatttaa ag#taaatgct    300ttgagtggag gaactcaagt agcagcagga gcaggtttgg aagcagttaa ag#aaagtgga    360ggacaaggaa aaagttatct attgggaact tctgcttcta tcaacttagt ga#acaatgaa    420gtttctgcaa aatcagaaaa taatacagta gcaggagaat ctgaaagcca aa#aaatggat    480gttgatgtca ctgcttatca agcggacacc caagtgacag gagctttaaa tt#tacaagct    540ggaaagtcaa atggaactgt aggggctact gtgactgttg ccaaattaaa ca#acaaagta    600aatgcttcta ttagtggtgg gagatatact aacgttaatc gagcggacgc aa#aagctctt    660ttagcaacca ctcaagtgac tgctgcagtg acgacgggag ggacaattag tt#ctggagcg    720ggattaggaa attatcaagg ggctgtttct gtcaataaga ttgacaatga cg#tggaagct    780agcgttgata aatcttccat cgaaggagct aatgaaatca atgtcattgc ca#aagatgtc    840aaaggaagtt ctgatctagc aaaagaatat caggctttac taaatggaaa ag#ataaaaaa    900tatttagaag atcgtggtat taatacgact ggaaatggtt attatacgaa gg#aacaacta    960gaaaaagcaa agaaaaaaga aggagcggtc attgtaaatg ctgctttatc gg#ttgctgga   1020acggataaat ccgctggagg agtagctatt gcagtcaata ctgttaaaaa ta#aatttaaa   1080gcagaattga gtggaagcaa taaggaagcc ggagaggata aaattcatgc ga#aacatgta   1140aatgtggagg caaaatcatc tactgttgtt gtgaatgcgg cttctggact tg#ctatcagc   1200aaagatgctt tttcaggaat gggatctgga gcatggcaag acttatcaaa tg#acacgatt   1260gcaaaggtgg ataaaggaag aatttctgct gattccttaa atgtgaacgc aa#ataattcc   1320attcttgggg tgaatgttgc gggaaccatt gccggttctc tttctacggc gg#taggagct   1380gcttttgcga ataatactct tcataataaa acctctgctt tgattacagg aa#cgaaggta   1440aatcctttta gtggaaagaa tacaaaagtc aatgtacaag ctttgaatga tt#ctcatatt   1500acaaacgttt ctgctggagg cgctgcaagt attaagcagg ctggaatcgg ag#gaatggta   1560tctgtcaatc gtggttctga tgaaacggaa gctttagtta gtgattctga gt#ttgaagga   1620gtaagttctt tcaatgtaga tgcaaaagat caaaaaacaa taaatacaat tg#ccggaaat   1680gcaaatggag gaaaagcggc tggagttgga gcaacagttg ctcatacaaa ta#ttggaaaa   1740caatcagtta tagctattgt aaaaaacagt aaaattacaa cggcgaatga tc#aagataga   1800aaaaatatca atgtgactgc aaaagattat actatgacca atactatagc ag#tcggagtt   1860ggaggagcaa aaggagcctc tgtgcaagga gcttctgcaa gtactacctt ga#ataagaca   1920gtttcttctc atgttgatca aactgatatt gacaaagatt tagaggaaga aa#ataatgga   1980aataaggaaa aggcaaatgt taatgttcta gctgaaaata cgagtcaagt gg#tcacaaat   2040gcgacagtgc tttccggagc aagtggacaa gctgcagtag gagctggagt ag#cagttaat   2100 aaaattacac aaaatacttc tgcacatata aaaaatagta c    #                   # 2141 <210> SEQ ID NO 12 <211> LENGTH: 1887<212> TYPE: DNA <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 12ctgcagtagg agctggagta gcagttaata aaattacaca aaatacttct gc#acatataa     60aaaatagtac tcaaaatgta cgaaatgctt tggtaaaaag caaatctcat tc#atctatta    120aaacaattgg aattggagct ggagttggag ctggaggagc tggagtgaca gg#ttctgtag    180cagtgaataa gattgtaaat aatacgatag cagaattaaa tcatgcaaaa at#cactgcga    240agggaaatgt cggagttatt acagagtctg atgcggtaat tgctaattat gc#aggaacag    300tgtctggagt ggcccgtgca gcaataggag cctcaaccag tgtgaatgaa at#tacaggat    360ctacaaaagc atatgtaaaa gattctacag tgattgctaa agaagaaaca ga#tgattata    420ttactactca agggcaagta gataaagtgg tagataaagt attcaaaaat ct#taatatta    480acgaagactt atcacaaaaa agaaaaataa gtaataaaaa aggatttgtt ac#caatagtt    540cagctactca tactttaaaa tctttattgg caaatgccgc tggttcagga ca#agccggag    600tggcaggaac tgttaatatc aacaaggttt atggagaaac agaagctctt gt#agaaaatt    660ctatattaaa tgcaaaacat tattctgtaa aatcaggaga ttacacgaat tc#aatcggag    720tagtaggttc tgttggtgtt ggtggaaatg taggagtagg agcttcttct ga#taccaata    780ttataaaaag aaataccaag acaagagttg gaaaaactac aatgtctgat ga#aggtttcg    840gagaagaagc tgaaattaca gcagattcta agcaaggaat ttcctctttt gg#agtcggag    900tcgcagcagc cggggtagga gccggagtgg caggaaccgt ttccgtaaat ca#atttgcag    960gaaagacgga agtagatgtg gaagaagcaa agattttggt aaaaaaagct ga#gattacag   1020caaaacgtta tagttctgtt gcaattggaa atgccgcagt cggagtggct gc#aaaaggag   1080ctggaattgg agcagcagtg gcagttacca aagatgaatc aaacacgaga gc#aagagtga   1140aaaattctaa aattatgact cgaaacaagt tagatgtaat agcagaaaat ga#gataaaat   1200caggtactgg aatcggttca gccggagctg gaattcttgc agccggagta tc#tggagtgg   1260tttctgtcaa taatattgca aataaggtag aaacagatat cgatcatagt ac#tttacact   1320cttctactga tgtaaatgta aaagctctta ataaaatttc gaattccttg ac#agccggtg   1380gaggagccgc aggtcttgca gcagttaccg gagtggtttc tgttaacact at#aaatagtt   1440ctgtgatagc tcgagttcac aataactctg atttgacttc cgtacgagaa aa#agtaaatg   1500taacggcaaa agaggaaaaa aatattaagc aaacagcagc aaatgcagga at#cggaggag   1560cagcaatcgg agccaatgtc ttggtaaata attttggaac agctgtagaa ga#tagaaaaa   1620attctgaagg aaaaggaaca gaagttttaa aaactttaga cgaagttaac aa#agaacaag   1680ataaaaaagt aaatgatgct acgaaaaaaa tcttacaatc agcaggtatt tc#tacagaag   1740atacttctgt aaaagcggat agaggagata ctcagggaga aggaattaaa gc#cattgtga   1800agacttctga tattattgga aaaaatgtag atattacaac agaggacaag aa#taatatca   1860 cttctactgg tggtttggga actgcag          #                   #           1887 <210> SEQ ID NO 13<211> LENGTH: 2322 <212> TYPE: DNA<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 13ggaattaaag ccattgtgaa gacttctgat attattggaa aaaatgtaga ta#ttacaaca     60gaggacaaga ataatatcac ttctactggt ggtttgggaa ctgcaggtct tg#cttccgca    120tcaggaacag tggcagttac aaatattaaa agaaattccg gagttactgt tg#aaaattct    180tttgtgaaag cagctgaaaa agtaaatgtt agatcggata ttacaggaaa tg#ttgcttta    240acagcatatc aaggtcctgt aggagcattg ggaataggag ctgcctatgc ag#aattaaat    300tctaatggaa gatcaaatat cagtattaaa aattctaagc tattaggaaa aa#atattgat    360gttattgtaa aagataaatc ggaattgaga gcggaagcaa aaggattaac cg#taggagcg    420gtagctgccg gagccattat ctcaaaagca aagaatgaaa tgaattcaga gg#ttgaaatt    480gagaagagta ttttcaatga agaaaataga gtaactagcc cttctaaagg aa#ttggaaga    540gaaatcaatg tcaaagtgga aaaagaaaac agagtgactg ctgaatctca ag#gagcttct    600gtaggagcag tagcaggggc aggaattatt tccgaagcaa aagatgccgg aa#gctcttat    660ttgaaagtta gtacaaaatc cggaagaagt atttttcatg cagataatgt ga#atatggaa    720gcaacacata aaatgaaagt aacagcagtt tctaaagcag taacaggttc tg#tattggga    780ggagttggag tcaccaaggc agaagctact gctgcaggta aaactatggt ag#aagttgag    840gaaggaaatt tgttcagaac aaatcgattg aatgcaattt ctaaagtaga ag#gtttggat    900gaagataaag taactgctaa atcttctgta gtatcaggaa atggaggagg aa#ttgccgga    960gcaggagtga atacttctac agcacaaagt aatactgaat ccgtagttcg tt#tacgaaag   1020caagattatg aaaataatga ttacacaaaa aaatatattt cagaagtcaa tg#ctcttgct   1080ttaaatgata caaagaatga agcgaatata gaatctttag cggtagccgg tg#tgcatgca   1140caaggaacaa acaaagcatt tacgagatca aacaagttaa cttctacaac tg#taaatgga   1200ggaaacgtat ctcaacttcg tgcaaaagct ttggctaaaa atgaaaatta tg#gaaatgta   1260aaaggaactg gaggagcctt agtcggagcg gaaacagcag ccgttgaaaa tt#atacaaag   1320agtactacag gagcattggt tgcaggaaat tgggaaattg gagataaatt ag#aaacgatt   1380gcaagagata atacgattgt aagagtcaac ggagacggaa ccaaaggagg tc#ttgtcgga   1440aagaatggta tttctgtgaa aaatacaatt tcaggggaaa caaaatcatc ca#ttgaagat   1500aaagccagaa ttgttggaac cggaagtgta aatgtagatg ctttgaatga ac#ttgatgta   1560gatctacaag gaaaaagtgg tggctatggt ggaattggta ttggaaatgt tg#atgtaaat   1620aatgtgatta agaaaaatgt agaagccaaa atcggaagac atgctattgt ag#aaactact   1680ggaaaacaag aatatcaagc atttacaaga gcaaaagtaa atattcttgg aa#aaggagac   1740gctgcagctg cagctgcaat atcgaatgta cacatttcca atgagatgga ta#ttaaaaat   1800ttggcaaagc agtatgcatc ttctcaatta ataaccaaaa attcaaaaaa ta#atattact   1860ttagcatcaa gtagtgaatc gaatgtgaat gttcatgggg tggctgaagc aa#gaggtgca   1920ggagccaaag cgacagttag tgtaaagaat caaataaata gaactaataa tg#ttgattta   1980gcaggaaaaa ttaaaacaga gggaaacatc aatgtatatg ccggatatga ta#aaaattat   2040aatataagta agacaaattc taaggctatt gcggatgcca aaagtcatgc tg#cagctgct   2100tcggcaactg ccactattga aaaaaatgaa gtaaaattta ataatgcgat cc#gagaattt   2160aaaaataatc tggcaagatt ggaagggaaa gctaataaaa aaacgtcggt ag#gatctaat   2220caggtagact ggtatacgga taaatataca tggcattctt ctgaaaaagc at#acaaaaaa   2280 ttgacatatc aatcaaagag aggagaaaaa gggaaaaaat ga    #                   #2322 <210> SEQ ID NO 14 <211> LENGTH: 1017<212> TYPE: DNA <213> ORGANISM: Fusobacterium necrophorum<400> SEQUENCE: 14atcaatatgg cttccggaaa agttccggga acgaccgatt attttgtgca aa#tctatgaa     60ccaaaaagac agcagttttt tgtttttgca gataatttag gacaaaaaaa ta#caggagaa    120ttacgatggg ggctaaatta tattaataat agtgttacag gaaacagaga tc#aactgtct    180cttacctctt tagtaacaga aggaacggct tctctatctt ctttttatac tt#ttcctgtt    240tctaaaaaag gaaccaaaat atcactacaa cattctgtag gaaagttgaa ac#atatacaa    300ggggctttaa agcataaaat aactggaaac tcttatagtt atggggttgg aa#tagttcat    360cctattctgg ttcatgaaaa aaataaagta gaactttcct tggattgggt aa#aacaaagg    420actgttacag atctattgaa attgaaatgg gtaaataata gactttctaa gt#atacagcg    480ggaattggaa taagccatta tgaggaagat agtgttttct atacaaagca aa#atattaca    540aagggaaaat ttattccaat ttcgggagat gcaagaaatt atacaaagta tg#atatgttt    600ctaatatatc agaaaaactt gaaatataac actttagtaa cactaaagat gg#cagggcaa    660tattctctga gtaaaaaatt accctctgtc gagcaaattt atgcaggagg ag#cctataat    720gttcgtggtt atccggaaaa ttttatggga gctgaacacg gagttttttt ca#atgctgaa    780ttatcaaaat tagtagagaa taaaggagaa ttttttgttt ttttagatgg gg#cttctctt    840catggagaga gtgcttggca ggaaaataga atttttagct caggttttgg at#ataaaata    900aggtttttag aaaaaaataa tattgctgtt agcatggcat ttccatggaa ga#aaaaaata    960aatagtattt cagtagattc taatcgaatc tatattacaa taaatcatga at#tttaa      1017 <210> SEQ ID NO 15 <211> LENGTH: 11130 <212> TYPE: DNA<213> ORGANISM: Fusobacterium necrophorum <400> SEQUENCE: 15gatcaatatg gcttccggaa aagttccggg aacgaccgat tattttgtgc aa#atctatga     60accaaaaaga cagcagtttt ttgtttttgc agataattta ggacaaaaaa at#acaggaga    120attacgatgg gggctaaatt atattaataa tagtgttaca ggaaacagag at#caactgtc    180tcttacctct ttagtaacag aaggaacggc ttctctatct tctttttata ct#tttcctgt    240ttctaaaaaa ggaaccaaaa tatcactaca acattctgta ggaaagttga aa#catataca    300aggggcttta aagcataaaa taactggaaa ctcttatagt tatggggttg ga#atagttca    360tcctattctg gttcatgaaa aaaataaagt agaactttcc ttggattggg ta#aaacaaag    420gactgttaca gatctattga aattgaaatg ggtaaataat agactttcta ag#tatacagc    480gggaattgga ataagccatt atgaggaaga tagtgttttc tatacaaagc aa#aatattac    540aaagggaaaa tttattccaa tttcgggaga tgcaagaaat tatacaaagt at#gatatgtt    600tctaatatat cagaaaaact tgaaatataa cactttagta acactaaaga tg#gcagggca    660atattctctg agtaaaaaat taccctctgt cgagcaaatt tatgcaggag ga#gcctataa    720tgttcgtggt tatccggaaa attttatggg agctgaacac ggagtttttt tc#aatgctga    780attatcaaaa ttagtagaga ataaaggaga attttttgtt tttttagatg gg#gcttctct    840tcatggagag agtgcttggc aggaaaatag aatttttagc tcaggttttg ga#tataaaat    900aaggttttta gaaaaaaata atattgctgt tagcatggca tttccatgga ag#aaaaaaat    960aaatagtatt tcagtagatt ctaatcgaat ctatattaca ataaatcatg aa#ttttaaag   1020ggggtaagac aaaatgagcg gcatcaaaaa taacgttcag aggacaagga ag#aggatatc   1080agattctaaa aaagttttaa tgattttggg attgttgatt aacactatga cg#gtgagggc   1140taatgataca atcaccgcga ctgagaattt tggaacaaaa atagaaaaaa ag#gataatgt   1200ttatgacatt actacaaaca agattcaagg ggagaacgct tttaacagtt tt#aatagatt   1260tgctttaaca gaaaataata tagcaaatct atattttggg gaaaagaata gt#acgggggt   1320aaataatctt tttaactttg tcaatggaaa aattgaagta gatgggatta tc#aacggaat   1380tcgagaaaat aaaattggag gaaatttata tttcttaagc tcggaaggga tg#gcagtagg   1440aaaaaatgga gttatcaatg ctggttcttt tcattctatt attccaaaac aa#gatgattt   1500taagaaggct ttggaagaag ccaaacatgg taaagttttt aatggaatca tt#ccagtaga   1560tggaaaagta aaaattccat tgaatccgaa tggaagcatt acggtagaag ga#aaaatcaa   1620tgctgttgaa ggcatcggtt tatatgcggc ggatattaga ttgaaagata ct#gcaatact   1680aaagacagga attacagatt ttaaaaattt agtcaatatt agtgatcgaa ta#aattctgg   1740tctgaccgga gatttaaaag ctaccaagac aaaatctgga gatattattc tt#tcagctca   1800catagattct cctcaaaaag ctatgggaaa aaattcaact gttggaaaga ga#atagaaga   1860atatgtaaaa ggaaatacca aagcaaatat tgaatctgat gctgtattgg aa#gcagatgg   1920aaatataaaa attagtgcga aagctacaaa tgggagattt ataaagaaag aa#ggggaaaa   1980agaaacttat aacactcctt taagtttatc agatgtggaa gcttccgtaa ga#gtaaataa   2040aggaaaagtc ataggaaaga atgttgacat tacagctgaa gcaaagaatt tc#tatgatgc   2100aactttagtt actaagcttg caaagcactc ttttagcttt gttacaggtt ct#atttctcc   2160tatcaattta aatggatttt taggtttatt gacaagtaag tccagtgtcg tt#attggaaa   2220agatgccaaa gtcgaagcaa cagaaggaaa ggcaaatatt cattcttaca gt#ggagtaag   2280agcaactatg ggagcagcta cttctccatt aaaaattacc aatttatatt tg#gagaaagc   2340caatggaaaa cttctcagta tcggagcggg atatatttct gcaaaaagta at#tccaatgt   2400aactattgaa ggagaagtaa aatcgaaggg aagagcagat attacttcaa aa#tctgaaaa   2460tactattgat gcttctgttt ctgttggaac gatgagagat tccaataaag ta#gctctttc   2520agtattggtg acggaaggag aaaataaatc ttccgtcaag attgctaaag ga#gcaaaagt   2580agaatcagaa acggatgatg taaatgtgag aagtgaagcg attaattcca tt#cgagctgc   2640tgtaaaaggt ggattggggg atagtggtaa tggggttgtg gctgcaaata tt#tctaacta   2700taatgcttcc tcccgtatag atgtagatgg atatctacat gccaagaagc ga#ctaaatgt   2760ggaggctcat aacattacta aaaatagtgt tctgcaaaca ggatctgatt tg#ggaacttc   2820caagtttatg aatgatcacg tttatgaatc aggtcatcta aaatcaattt ta#gatgcaat   2880aaaacagcgg tttggaggag acagtgtcaa tgaggaaata aagaataagc ta#acgaactt   2940atttagtgtc ggtgtgtctg caaccatagc aaatcataat aattctgctt ct#gtggcaat   3000aggagagagt ggaagacttt cttcaggagt ggaagggagt aatgtaaggg ca#ttaaatga   3060agctcaaaat cttcgagcga ctacgtcaag tggaagtgtg gctgtacgaa ag#gaagaaaa   3120aaagaaactt attggaaatg cagcagtttt ttatggaaac tataaaaata at#gcttctgt   3180gacaattgcc gatcatgctg aattggtatc ggaaggaaaa attgatatca ac#agtgaaaa   3240taaaattgaa tataaaaatc cttcaaaaat ggcaaagtct gttattgata aa#ttagaact   3300tttaaagaga gcttttggaa aagaaacgaa aactccagaa tatgatccga aa#gatattga   3360atctattgaa aaattattga atgcattttc agaaaaattg gatggaaaac cg#gagctttt   3420actaaatggt gaaagaatga caattattct tccggatgga acttcaaaaa ca#ggaactgc   3480tatagaaatt gcaaactatg ttcagggaga aatgaaaaaa ttagaggaaa aa#ttaccgaa   3540aggatttaaa gctttttcag aaggattgag tggactgatt aaagaaactt tg#aattttac   3600aggagtagga aattatgcaa attttcacac ttttacctct tccggagcta at#ggagaaag   3660agatgtttct tctgtgggag gagctgtttc gtgggtagaa caggagaatt at#agcaaggt   3720atccgttgga aaaggagcta aacttgctgc aaaaaaagat ttaaatataa aa#gctatcaa   3780taaagcagaa acagtgaatt tagttggaaa tattggactt gcgagaagca gt#acatccgg   3840aagtgcagtc ggaggaagat taaatgttca aagatcgaaa aattcagcta tc#gtagaagc   3900taaagaaaaa gctgaattat caggagaaaa tattaatgca gatgcattga ac#agactttt   3960tcatgtagcg ggatctttta atggtggctc aggtgggaat gcaatcaatg ga#atgggaag   4020ttatagtgga ggtatcagta aggcaagagt ttccattgat gacgaagcat at#ttgaaagc   4080taataaaaaa attgctttaa acagtaagaa tgatacttct gtttggaatg ct#gccggttc   4140agcgggaatc ggaacgaaaa atgcggcggt cggggttgct gttgcggtaa at#gattatga   4200tatttcaaac aaagcttcca ttgaagataa tgacgaagga caaagtaaat at#gataagaa   4260taaagatgat gaagtaacag taactgcgga atctttagaa gtagatgcaa aa#acgaccgg   4320aacaatcaac agtatttctg ttgccggagg aattaataag gttggaagta aa#ccgagtga   4380agaaaaaccg aaatcagaag aaagaccaga gggatttttt ggcaaaatcg ga#aacaaagt   4440ggactctgta aaaaataaaa ttacggatag tatggattca ttaacagaaa aa#attacaaa   4500ttacatttct gaaggagtaa aaaaagcggg gaatcttcct tcgaacgttt ct#catactcc   4560cgataaagga ccgtctttca gtttgggagc ttctggaagt gtttctttca at#aatattaa   4620aaaggaaaca tctgctgtcg tagatggagt aaagataaat ttgaagggag ca#aataaaaa   4680ggtagaggtg acttcttctg attctacttt tgttggagca tggggcggat ct#gctgcact   4740tcagtggaat catattggaa gtggaaatag caacatcagt gctggtttag ct#ggagcggc   4800tgctgtaaat aatattcaaa gtaaaacaag tgctttggtt aaaaatagtg at#attcgaaa   4860tgccaataaa tttaaagtaa atgctttgag tggaggaact caagtagcag ca#ggagcagg   4920tttggaagca gttaaagaaa gtggaggaca aggaaaaagt tatctattgg ga#acttctgc   4980ttctatcaac ttagtgaaca atgaagtttc tgcaaaatca gaaaataata ca#gtagcagg   5040agaatctgaa agccaaaaaa tggatgttga tgtcactgct tatcaagcgg ac#acccaagt   5100gacaggagct ttaaatttac aagctggaaa gtcaaatgga actgtagggg ct#actgtgac   5160tgttgccaaa ttaaacaaca aagtaaatgc ttctattagt ggtgggagat at#actaacgt   5220taatcgagcg gacgcaaaag ctcttttagc aaccactcaa gtgactgctg ca#gtgacgac   5280gggagggaca attagttctg gagcgggatt aggaaattat caaggggctg tt#tctgtcaa   5340taagattgac aatgacgtgg aagctagcgt tgataaatct tccatcgaag ga#gctaatga   5400aatcaatgtc attgccaaag atgtcaaagg aagttctgat ctagcaaaag aa#tatcaggc   5460tttactaaat ggaaaagata aaaaatattt agaagatcgt ggtattaata cg#actggaaa   5520tggttattat acgaaggaac aactagaaaa agcaaagaaa aaagaaggag cg#gtcattgt   5580aaatgctgct ttatcggttg ctggaacgga taaatccgct ggaggagtag ct#attgcagt   5640caatactgtt aaaaataaat ttaaagcaga attgagtgga agcaataagg aa#gccggaga   5700ggataaaatt catgcgaaac atgtaaatgt ggaggcaaaa tcatctactg tt#gttgtgaa   5760tgcggcttct ggacttgcta tcagcaaaga tgctttttca ggaatgggat ct#ggagcatg   5820gcaagactta tcaaatgaca cgattgcaaa ggtggataaa ggaagaattt ct#gctgattc   5880cttaaatgtg aacgcaaata attccattct tggggtgaat gttgcgggaa cc#attgccgg   5940ttctctttct acggcggtag gagctgcttt tgcgaataat actcttcata at#aaaacctc   6000tgctttgatt acaggaacga aggtaaatcc ttttagtgga aagaatacaa aa#gtcaatgt   6060acaagctttg aatgattctc atattacaaa cgtttctgct ggaggcgctg ca#agtattaa   6120gcaggctgga atcggaggaa tggtatctgt caatcgtggt tctgatgaaa cg#gaagcttt   6180agttagtgat tctgagtttg aaggagtaag ttctttcaat gtagatgcaa aa#gatcaaaa   6240aacaataaat acaattgccg gaaatgcaaa tggaggaaaa gcggctggag tt#ggagcaac   6300agttgctcat acaaatattg gaaaacaatc agttatagct attgtaaaaa ac#agtaaaat   6360tacaacggcg aatgatcaag atagaaaaaa tatcaatgtg actgcaaaag at#tatactat   6420gaccaatact atagcagtcg gagttggagg agcaaaagga gcctctgtgc aa#ggagcttc   6480tgcaagtact accttgaata agacagtttc ttctcatgtt gatcaaactg at#attgacaa   6540agatttagag gaagaaaata atggaaataa ggaaaaggca aatgttaatg tt#ctagctga   6600aaatacgagt caagtggtca caaatgcgac agtgctttcc ggagcaagtg ga#caagctgc   6660agtaggagct ggagtagcag ttaataaaat tacacaaaat acttctgcac at#ataaaaaa   6720tagtactcaa aatgtacgaa atgctttggt aaaaagcaaa tctcattcat ct#attaaaac   6780aattggaatt ggagctggag ttggagctgg aggagctgga gtgacaggtt ct#gtagcagt   6840gaataagatt gtaaataata cgatagcaga attaaatcat gcaaaaatca ct#gcgaaggg   6900aaatgtcgga gttattacag agtctgatgc ggtaattgct aattatgcag ga#acagtgtc   6960tggagtggcc cgtgcagcaa taggagcctc aaccagtgtg aatgaaatta ca#ggatctac   7020aaaagcatat gtaaaagatt ctacagtgat tgctaaagaa gaaacagatg at#tatattac   7080tactcaaggg caagtagata aagtggtaga taaagtattc aaaaatctta at#attaacga   7140agacttatca caaaaaagaa aaataagtaa taaaaaagga tttgttacca at#agttcagc   7200tactcatact ttaaaatctt tattggcaaa tgccgctggt tcaggacaag cc#ggagtggc   7260aggaactgtt aatatcaaca aggtttatgg agaaacagaa gctcttgtag aa#aattctat   7320attaaatgca aaacattatt ctgtaaaatc aggagattac acgaattcaa tc#ggagtagt   7380aggttctgtt ggtgttggtg gaaatgtagg agtaggagct tcttctgata cc#aatattat   7440aaaaagaaat accaagacaa gagttggaaa aactacaatg tctgatgaag gt#ttcggaga   7500agaagctgaa attacagcag attctaagca aggaatttcc tcttttggag tc#ggagtcgc   7560agcagccggg gtaggagccg gagtggcagg aaccgtttcc gtaaatcaat tt#gcaggaaa   7620gacggaagta gatgtggaag aagcaaagat tttggtaaaa aaagctgaga tt#acagcaaa   7680acgttatagt tctgttgcaa ttggaaatgc cgcagtcgga gtggctgcaa aa#ggagctgg   7740aattggagca gcagtggcag ttaccaaaga tgaatcaaac acgagagcaa ga#gtgaaaaa   7800ttctaaaatt atgactcgaa acaagttaga tgtaatagca gaaaatgaga ta#aaatcagg   7860tactggaatc ggttcagccg gagctggaat tcttgcagcc ggagtatctg ga#gtggtttc   7920tgtcaataat attgcaaata aggtagaaac agatatcgat catagtactt ta#cactcttc   7980tactgatgta aatgtaaaag ctcttaataa aatttcgaat tccttgacag cc#ggtggagg   8040agccgcaggt cttgcagcag ttaccggagt ggtttctgtt aacactataa at#agttctgt   8100gatagctcga gttcacaata actctgattt gacttccgta cgagaaaaag ta#aatgtaac   8160ggcaaaagag gaaaaaaata ttaagcaaac agcagcaaat gcaggaatcg ga#ggagcagc   8220aatcggagcc aatgtcttgg taaataattt tggaacagct gtagaagata ga#aaaaattc   8280tgaaggaaaa ggaacagaag ttttaaaaac tttagacgaa gttaacaaag aa#caagataa   8340aaaagtaaat gatgctacga aaaaaatctt acaatcagca ggtatttcta ca#gaagatac   8400ttctgtaaaa gcggatagag gagatactca gggagaagga attaaagcca tt#gtgaagac   8460ttctgatatt attggaaaaa atgtagatat tacaacagag gacaagaata at#atcacttc   8520tactggtggt ttgggaactg caggtcttgc ttccgcatca ggaacagtgg ca#gttacaaa   8580tattaaaaga aattccggag ttactgttga aaattctttt gtgaaagcag ct#gaaaaagt   8640aaatgttaga tcggatatta caggaaatgt tgctttaaca gcatatcaag gt#cctgtagg   8700agcattggga ataggagctg cctatgcaga attaaattct aatggaagat ca#aatatcag   8760tattaaaaat tctaagctat taggaaaaaa tattgatgtt attgtaaaag at#aaatcgga   8820attgagagcg gaagcaaaag gattaaccgt aggagcggta gctgccggag cc#attatctc   8880aaaagcaaag aatgaaatga attcagaggt tgaaattgag aagagtattt tc#aatgaaga   8940aaatagagta actagccctt ctaaaggaat tggaagagaa atcaatgtca aa#gtggaaaa   9000agaaaacaga gtgactgctg aatctcaagg agcttctgta ggagcagtag ca#ggggcagg   9060aattatttcc gaagcaaaag atgccggaag ctcttatttg aaagttagta ca#aaatccgg   9120aagaagtatt tttcatgcag ataatgtgaa tatggaagca acacataaaa tg#aaagtaac   9180agcagtttct aaagcagtaa caggttctgt attgggagga gttggagtca cc#aaggcaga   9240agctactgct gcaggtaaaa ctatggtaga agttgaggaa ggaaatttgt tc#agaacaaa   9300tcgattgaat gcaatttcta aagtagaagg tttggatgaa gataaagtaa ct#gctaaatc   9360ttctgtagta tcaggaaatg gaggaggaat tgccggagca ggagtgaata ct#tctacagc   9420acaaagtaat actgaatccg tagttcgttt acgaaagcaa gattatgaaa at#aatgatta   9480cacaaaaaaa tatatttcag aagtcaatgc tcttgcttta aatgatacaa ag#aatgaagc   9540gaatatagaa tctttagcgg tagccggtgt gcatgcacaa ggaacaaaca aa#gcatttac   9600gagatcaaac aagttaactt ctacaactgt aaatggagga aacgtatctc aa#cttcgtgc   9660aaaagctttg gctaaaaatg aaaattatgg aaatgtaaaa ggaactggag ga#gccttagt   9720cggagcggaa acagcagccg ttgaaaatta tacaaagagt actacaggag ca#ttggttgc   9780aggaaattgg gaaattggag ataaattaga aacgattgca agagataata cg#attgtaag   9840agtcaacgga gacggaacca aaggaggtct tgtcggaaag aatggtattt ct#gtgaaaaa   9900tacaatttca ggggaaacaa aatcatccat tgaagataaa gccagaattg tt#ggaaccgg   9960aagtgtaaat gtagatgctt tgaatgaact tgatgtagat ctacaaggaa aa#agtggtgg  10020ctatggtgga attggtattg gaaatgttga tgtaaataat gtgattaaga aa#aatgtaga  10080agccaaaatc ggaagacatg ctattgtaga aactactgga aaacaagaat at#caagcatt  10140tacaagagca aaagtaaata ttcttggaaa aggagacgct gcagctgcag ct#gcaatatc  10200gaatgtacac atttccaatg agatggatat taaaaatttg gcaaagcagt at#gcatcttc  10260tcaattaata accaaaaatt caaaaaataa tattacttta gcatcaagta gt#gaatcgaa  10320tgtgaatgtt catggggtgg ctgaagcaag aggtgcagga gccaaagcga ca#gttagtgt  10380aaagaatcaa ataaatagaa ctaataatgt tgatttagca ggaaaaatta aa#acagaggg  10440aaacatcaat gtatatgccg gatatgataa aaattataat ataagtaaga ca#aattctaa  10500ggctattgcg gatgccaaaa gtcatgctgc agctgcttcg gcaactgcca ct#attgaaaa  10560aaatgaagta aaatttaata atgcgatccg agaatttaaa aataatctgg ca#agattgga  10620agggaaagct aataaaaaaa cgtcggtagg atctaatcag gtagactggt at#acggataa  10680atatacatgg cattcttctg aaaaagcata caaaaaattg acatatcaat ca#aagagagg  10740agaaaaaggg aaaaaatgaa tttaagagag agtaaattta gtgagttttt aa#aaaattca  10800aacataactt gttttgaaag agaagaagtg aaagatgagt tagaaacagt tg#tatatcga  10860agttttatgg aagtagaggg acaaaattta cctatggtaa ttgtgatgga ta#acagtatt  10920tatacgaata tccgagtgca aattgctcca aaagtcataa aagatactaa ta#aagaagcg  10980gtactttcct atatcaatga attgaaccga gaatacaaag tatttaaata tt#atgtgaca  11040gaggatgcag atgtttgttt agatagttgt gtaacctcca ttgcagaaga at#ttaatcca  11100 gaaatggttt acactatttt aaatgtgatc         #                   #        11130

We claim:
 1. An isolated polypeptide consisting of SEQ ID No.
 2. 2. Avaccine comprising the isolated peptide of SEQ ID No. 2 and apharmacologically acceptable carrier.
 3. A recombinantly derived F.necrophorum polypeptide consisting of SEQ ID No.
 2. 4. A vaccinecomprising the recombinant polypeptide of claim 3 and apharmacologically acceptable carrier.